Patent Application
20230227508
Protein-protein interactions play an important role in elucidating the mechanisms of biological systems and in numerous clinical applications. For example, during viral infection, viral surface proteins bind to host cell receptors to promote internalization of the viral genome. Inhibitors of the interaction between a viral surface protein and host cell receptors may be used to prevent viral infection or spread of such an infection to other host cells. Elucidation of protein-protein interactions have also led to development of immunotherapies and antibodies, which has been useful in the treatment of cancer. Accordingly, efficient methods of identifying peptide binders of target proteins are warranted.
Aspects of the present disclosure relate to peptides binders of target proteins that may be useful in the treatment of disease and methods of identifying such peptides. Further aspects of the present disclosure provide non-naturally occurring peptides. In some embodiments, a non-naturally occurring peptide comprise:
In some embodiments, the non-naturally occurring peptide comprises scaffold L5 and a sequence selected from SEQ ID NOS: 6-16; and/or scaffold L3 and a sequence selected from SEQ ID NOs: 17-25. In some embodiments, the non-naturally occurring peptide comprises scaffold L3 and SEQ ID NO: 24.
Further aspects of the present disclosure provide host cells comprising a heterologous nucleic acid encoding any of the non-naturally occurring peptides disclosed herein.
In some embodiments, the heterologous nucleic acid further encodes SEQ ID NO: 46.
In some embodiments, the heterologous nucleic acid comprises any one of SEQ ID NOs: 47-66.
Further aspects of the present disclosure provide:
In some embodiments,
In some embodiments, the cell is a eukaryotic or prokaryotic cell, optionally wherein the prokaryotic cell is a bacterial cell.
In some embodiments, the transcription factor is a sigma factor (a factor).
In some embodiments, the first fusion protein is encoded by a first heterologous nucleic acid and the second fusion is encoded by a second heterologous nucleic acid.
In some embodiments, the candidate peptide comprises a sequence selected from SEQ ID NOs: 6-25 or comprises the non-naturally occurring peptide of any one of claims 1 or 2, optionally wherein the candidate peptide further comprises SEQ ID NO: 46.
In some embodiments, the at least one reporter gene encodes a positive selection marker, a negative selection marker, and/or a fluorescent protein, optionally wherein the positive selection marker is an antibiotic resistance gene, optionally wherein the antibiotic resistance gene is chloramphenicol acetyltransferase (cat), optionally wherein the negative selection marker is the herpes simplex virus-thymidine kinase (hsvtk) gene.
In some embodiments, the inducible promoter is an ECF promoter.
In some embodiments, the target protein comprises viral receptor binding domain (RBD) of the SARS-CoV-2 spike protein.
In some embodiments, the RBD comprises SEQ ID NO: 71.
In some embodiments, the host cells further comprises one or more enzymes selected from ProcM, LynD, TgnB, or PapB, optionally wherein the host cell comprises a heterologous nucleic acid encoding the enzyme, optionally wherein the heterologous nucleic acid encoding the enzyme comprises an inducible promoter.
Further aspects of the disclosure provide methods of identifying a peptide that binds a target protein. In some embodiments, the methods comprise culturing any of the host cells disclosed herein and detecting transcription of the at least one reporter gene, thereby identifying the candidate peptide as being capable of binding to the target protein.
In some embodiments, the methods comprise incubating in a reaction vessel:
detecting transcription of the reporter gene, thereby identifying the candidate peptide as being capable of binding to the target protein.
Further aspects of the present disclosure provide methods of treating a subject having or suspected of having a SARS-CoV-2 infection comprising administering an effective amount of any of the non-naturally occurring peptides disclosed herein.
In some embodiments, the method comprises repeating the method with a plurality of candidate peptides.
In some embodiments, culturing comprises positive and/or negative selection of the host cell.
In some embodiments, the method further comprises sequencing.
Further aspects of the disclosure provide libraries of peptides. In some embodiments, a library compress a plurality of peptides, wherein each peptide of the plurality of peptides has a length of n amino acids and has an amino acid sequence defined by a motif X1X2X3X4 . . . Xn, wherein n is 5-100, wherein each of X1-Xn is independently selected from a group consisting of up to 20 amino acids and at least one of X1-Xn within each peptide is an amino acid selected from a group consisting of 19 or fewer amino acids, and wherein the motif X1X2X3X4 . . . Xn is determined to be susceptible to post-translational modification by at least 2 distinct protein modification enzymes.
In some embodiments, less than 80% of the plurality of peptides are capable of being fully modified by the at least 2 distinct protein modification enzymes.
In some embodiments, at least one of X1-Xn is defined to be a single amino acid.
According to some aspects of the disclosure, compositions comprising host cells are provided. In some embodiments, a composition comprises a plurality of host cells, each host cell of the plurality comprising a peptide of a library disclosed herein, wherein the peptide comprised by each host cell is independent of the peptide comprised by each other host cell. In some embodiments, the composition comprises each peptide of the plurality of peptides. In some embodiments, the host cells are bacterial cells. In some embodiments, the peptide is encoded by a first nucleic acid sequence in the host cell. In some embodiments, each host cell further comprises at least one protein modifying enzyme. In some embodiments, the at least one protein modifying enzyme is encoded by a second nucleic acid sequence in the host cell.
Further aspects of the disclosure provide methods of designing amino acid motifs. In some embodiments, a method of designing an amino acid motif comprises:
(i) selecting one or more protein modifying enzymes;
(ii) identifying a recognition site (RS) sequence for each of the one or more protein modifying enzymes;
(iii) constructing a plurality of nucleic acid molecules, each nucleic acid molecule encoding a leader amino acid sequence comprising the RS sequence for each of the one or more protein modifying enzymes;
(iv) assigning a score to each of the plurality of nucleic acid molecules; and
(v) selecting one of the plurality of nucleic acid molecules based on step (iv),
to design the amino acid motif, wherein each RS sequence facilitates interaction of the corresponding protein modifying enzyme to a peptide defined by the amino acid motif, and wherein the leader amino acid sequence encoded by the nucleic acid molecule selected in step (v) is comprised within each peptide defined by the amino acid motif.
In some embodiments, each peptide defined by the amino acid motif further comprises a core sequence.
In some embodiments, the core sequence comprises one or more amino acids susceptible to modification by the one or more protein modifying enzymes.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. For purposes of clarity, not every component may be labeled in every drawing. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure. In the drawings:
Aspects of the present disclosure provide efficient methods of identifying peptide binders of target proteins using an intein-based system. As shown herein, the method is useful in identifying peptide binders of a target protein, including the viral receptor binding domain (RBD) of spike protein from SARS-CoV-2. In some embodiments, the methods disclosed herein have been used to identify modified peptide binders of RBD. Additional methods disclosed herein provide an efficient means of identifying peptides with particular properties and/or activity, such as biological activity. Libraries of peptides with useful characteristics are also provided, in addition to methods for their preparation and screening.
Without being bound by a particular theory, modified peptide binders have numerous advantages over traditional drug candidates including small molecule compounds and monoclonal antibodies (mABs). For example, small molecule compounds are often poor inhibitors of macromolecular interactions due to the physicochemical constraints of small molecule compounds; small molecule compounds are often not large enough to cover large binding interfaces. While mABs may be capable of occupying a larger binding surface area as compared to small molecule compounds, development of mABs is often slow, often taking about six months to identify a lead mAB against a target protein, have low stability, often require particular routes of administration (e.g., parenteral administration), and may have low cell penetrability. The methods and modified peptides described herein, in some embodiments, overcome many of these limitations. For example, in some embodiments, the peptide binders comprise modifications that increase stability, promote proteolytic resistance, and/or increase solubility.
Furthermore, conventional antibiotics used as drugs target diverse bacteria as part of their mode of action. This “broad-spectrum” activity has benefit in the treatment of life-threatening bacterial infections, as a single agent is able to address a large number of clinical indications. However, this broad-spectrum activity can also disrupt the subject's microbiome, leading to associated complications in health. The methods disclosed herein provide means for identifying peptides with antimicrobial activity, including narrow-spectrum activity. Narrow-spectrum antimicrobial agents are desirable to avoid microbiome disruptions and to mitigate selection pressure for widespread evolution of resistance to antibiotics. Narrow spectrum agents that can selectively remove specific bacteria are useful as both a subject-specific medicine, and as tool compounds to facilitate understanding of and manipulate the microbiome.
In early-stage drug discovery, candidate compounds are typically identified from two sources: natural products (e.g., isolated from natural sources such as plants or microbes) and combinatorial chemistry libraries of synthetic molecules. Inadequacies in ability to synthesize natural product-like molecules, as well as the prohibitive cost of identifying such molecules from nature, limit the ability to develop products (e.g., peptides) with desirable properties. In addition, molecules from combinatorial chemistry libraries lack the structural complexity necessary to identify ideal drug candidates. Engineered RiPPs provide the ability to biosynthesize structurally diverse small molecules (e.g., peptides) for screening and drug discovery.
In some embodiments, the methods disclosed herein allow for efficient methods of identifying candidate drugs against challenging therapeutic targets (e.g., targets that have been referred to as “undruggable”). Several cancer targets including KRAS, MYC, and transcription factors have been labeled as “undruggable targets” due to their large protein-protein interaction interfaces or due to the absence of protein pockets for binding. See, e.g., Whitfield et al., Front. Cell Dev. Biol. 5, 10 (2017) and McCormick et al., Clin. Cancer Res. 21, 1797-1801 (2015). In some embodiments, challenging therapeutic targets include particular microbes (e.g., drug-resistant bacteria, or bacteria of a class or species that is difficult to treat).
Aspects of the present disclosure provide methods of identifying peptide binders of a target protein using split intein-based selection system. Additional aspects of the present disclosure provide methods of identifying peptides with particular desired properties, such as biological activity using a split intein-based selection system.
An intein is an internal amino acid sequence that is post-translationally autoprocessed. During protein splicing, an intein self-excises from a precursor protein and ligates the flanking N- and C-terminal amino acid sequences (exteins or external protein sequences) via a new peptide bond. For example, a precursor protein may comprise the following configuration: N-extein-intein-C-extein. Following protein splicing, the following peptide is produced: N-extein-C-extein.
The intein, however, may be provided as two separate fragments (split inteins) rather than as contiguous sequence. During trans-splicing, the two fragments of the intein have to associate before protein splicing can occur. As used herein, an N-terminal intein (N-intein) comprises the N-terminal sequence of an intein, while the C-terminal intein (C-intein) comprises the C-terminal sequence of the same intein. When split inteins are used, the N-intein is linked to the C′ terminal end of the N-extein; the C-intein is located at the N′ end of the C-extein. The N-extein and the C-intein may belong to the same protein of interest. For example, the N-extein may comprise an N-terminal fragment of a protein of interest, while the C-intein comprises the C-terminal fragment of the same protein of interest, such that when the N-intein and C-intein associate, a full-length protein of interest is formed. See, e.g., Shah and Muir, Chem Sci. 2014; 5(1):446-461.
Any complementary split intein pair may be used including those known in the art. Non-limiting examples of complementary split inteins include the N-terminal intein NpuDNAE intein N (SEQ ID NO: 68) and the C-terminal intein NpuDNAE intein C (SEQ ID NO: 67). See also, e.g., US20200055900 and Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5.
In some embodiments, the methods described herein comprise using split inteins. In general, unless indicated otherwise, the split intein-based selection system described herein comprises two fusion proteins and an inducible promoter operably linked to a reporter gene. For example, the first fusion protein generally comprises (i) a first fragment of a transcription factor, (ii) a first split intein, and (iii) a target protein, and the second fusion protein may comprise (i) a candidate peptide, (ii) a second split intein, and (iii) a second fragment of the transcription factor. The first and second split inteins are complementary fragments, such that association of the first split intein with the second split intein promotes trans-splicing and formation of a full-length transcription factor to drive expression from the inducible promoter. As described below, it may also be possible to use the split intein-based system described herein without the need for a reporter gene operably linked to an inducible promoter (e.g., the fragments of the transcription factor may be replaced with fragments of a reporter protein).
In some embodiments, the first fusion protein comprises (i) a first fragment of a transcription factor, (ii) a first split intein, and (iii) a target protein linked sequentially from the N-terminus to the C-terminus, in which the first fragment is an N-terminal fragment of the transcription factor and the first split intein is an N-terminal split intein; and the second fusion comprises: (i) a candidate peptide, (ii) a second split intein, and (iii) a second fragment of the transcription factor linked sequentially from the N-terminus to the C-terminus, in which the second split intein is a C-terminal split intein, and the second fragment is a C-terminal fragment of the transcription factor.
In some embodiments, from the N-terminus to the C-terminus, the first fusion protein comprises a target protein linked to a first split intein linked to a first fragment of a transcription factor in which the first fragment is a C-terminal fragment of the transcription factor and the first split intein is a C-terminal split intein; and from the N-terminus to the C-terminus, the second fusion protein comprises a second fragment of the transcription factor linked to a second split intein linked to a candidate peptide, in which the second split intein is a N-terminal split intein and the second fragment is a N-terminal fragment of the transcription factor.
The first and second fusion proteins of the split intein-based selection system described herein may be used together with a nucleic acid comprising an inducible promoter operably linked to at least one reporter gene. Without being bound by a particular theory, binding of the (i) target protein in the first fusion protein with (ii) the candidate peptide in the second fusion protein brings the complementary split-intein in each fusion protein together to allow for protein splicing and release of a full-length transcription factor. The full-length transcription factor may then drive transcription from its cognate promoter. As used herein, a transcription factor is a protein that controls transcription (e.g., drives expression of a nucleic acid that is operably linked to a promoter). In some embodiments, a transcription factor binds to a promoter and drives transcription from the promoter. In some embodiments a transcription factor is an initiation factor. In some embodiments, a transcription factor is a sigma factor.
The promoter is operably linked to a reporter gene. A promoter is a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. A promoter is considered to be ‘operably linked’ to a nucleotide sequence when it is in a correct functional location and orientation in relation to the nucleotide sequence to control (‘drive’) transcriptional initiation and/or expression of that sequence. Promoters may be constitutive or inducible.
An inducible promoter is a promoter that is regulated (e.g., activated or inactivated) by the presence or absence of a particular factor. Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline responsive promoter systems, which include a tetracycline repressor protein, steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), pH-regulated promoters, and light-regulated promoters. A non-limiting example of an inducible system that uses a light-regulated promoter is provided in Wang et al., Nat. Methods. 2012 Feb. 12; 9(3):266-9.
Non-limiting examples of inducible promoters include the inducible T5 lacO promoter, which may be induced by Isopropyl β-d-1-thiogalactopyranoside (IPTG), pCym promoter, which may be induced by cumate and a sigma-factor sensitive promoter, including an extra-cytoplasmic function (ECF) promoter.
In some embodiments, the promoter operably linked to a reporter gene is an extra-cytoplasmic function (ECF) promoter and the transcription factor is a sigma factor. In some embodiments, a Sigma factor comprises the N-terminal sequence ECF20_992 N (SEQ ID NO: 70) and the C-terminal sequence ECF20_992 C (SEQ ID NO: 69). Initiation of transcription in bacteria requires a sigma factor (a factor or specificity factor). Sigma factors bind to bacterial RNA polymerase to form a holoenzyme and initiate transcription. Non-limiting examples of sigma factors include extracytoplasmic function (ECF) a factors, a70 (RpoD), a19 (FecI), a24 (RpoE), a28 (RpoF/FliA), a32 (RpoH), a38 (RpoS), and 654 (RpoN). In some embodiments, a sigma factor is not a housekeeping sigma factor. In some embodiments, a sigma factor that is used is not native to a host cell and allows for orthogonal gene expression. As a non-limiting example, a sigma factor from B. subtilis that is not naturally expressed in E. coli may be used in E. coli for orthogonal gene expression. See also, e.g., Bervoets et al., Nucleic Acids Res. 2018 Feb. 28; 46(4): 2133-2144 and Pinto et al., Nucleic Acids Res. 2018 Aug. 21; 46(14):7450-7464. As would be appreciated by one of ordinary skill in the art, a particular sigma factor may require particular promoter elements to promote transcription and/or a particular environmental trigger including, e.g., heat. In some embodiments, additional activator proteins may be required for a sigma factor to function.
Non-limiting examples of reporter genes include genes that encode fluorescent proteins, enzymes, and antibiotic resistance genes. A reporter gene may allow for positive or negative selection.
In some embodiments, a reporter gene encodes a selection marker, such as an antibiotic resistance gene (e.g., bsd, neo, hygB, pac, ble, or Sh bla) and/or a gene encoding a fluorescent protein (RFP, BFP, YFP, or GFP). In some embodiments, the antibiotic resistance gene is cat, which encodes chloramphenicol acetyltransferase. Cells may be selected for resistance to chloramphenicol by culturing the cells in the presence of chloramphenicol. In some embodiments, the selection marker enables selection of cells expressing a protein of interest (e.g., a full-length transcription factor). As would be appreciated by one of ordinary skill in the art, the effective amount of a selection agent may vary depending on the host cell and phenotype of interest.
Positive selection markers are selection markers that confer a selective advantage to a host cell. In some embodiments, positive selection is the use of such selection markers to confer a growth or survival advantage to a cell comprising a protein of interest. In some embodiments, positive selection is used to identify cells in which a candidate peptide binds a target protein. Without being bound by a particular theory, protein splicing of the fusion proteins in the split intein-based selection system disclosed herein is dependent on the association of the candidate peptide with the target protein; therefore, in the absence of a binding interaction or when the binding interaction is weak, expression of the reporter gene is low. In some embodiments, a candidate peptide binder of a target protein increases expression of the reporter gene in a host cell comprising the split intein-based selection system disclosed herein by at least 10%, at least 20%, at least 30%, at least 40%, at 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 100% relative to a control. In some embodiments, a control is a control peptide that has non-specific binding to the same target protein of interest. In some embodiments, a control is the level of expression of the candidate peptide binder in a host cell that comprises a split intein-based selection system with a control target protein that is not of interest.
Negative selection markers are selection markers that confer a selective disadvantage to a host cell. In some embodiments, negative selection is the use of such selection markers to confer a growth or survival disadvantage to a cell comprising an undesirable phenotype. Non-limiting examples of negative selection markers include Herpes Simplex Virus-1 Thymidine Kinase (HsvTK). Cells expressing HsvTK can be selected against by contacting cells with nucleotide 6-(β-D-2-deoxyribofuranosyl)-3,4-dihydro8H-pyrimido [4,5-c][1,2] oxazin-7-one (dP). Without being bound by a particular theory, expression of HsvTK alone without the addition of dP does not confer a growth disadvantage, which allows for temporal control of selection. As a non-limiting example, negative selection may be used to deplete host cells comprising candidate peptides that bind off-target proteins (identify candidates that non-specifically bind to a target protein of interest); the reporter gene may comprise a negative selection gene. For example, the split intein-based selection system described herein may be used with the candidate peptide and an off-target control protein in place of the target protein of interest to identify candidate peptides that bind to the off-target protein. In this embodiment, the inducible promoter may be operably linked to a gene encoding a negative selection marker and cells expressing the negative selection marker may be depleted by contacting the cells with the negative selection agent. Without being bound by a particular theory, the expression of the negative selection marker in this system is indicative of binding between the candidate peptide and the off-target control protein. In some embodiments, a reporter gene in the split intein-based selection system described herein comprises a negative selection marker to deplete cells that comprise an undesirable candidate peptide. As a non-limiting example, it may be desirable to select for peptide binders that specifically bind a target protein when the peptide is modified (e.g., comprising one or more post-translational modifications) but not when the peptide is unmodified. In some embodiments, the unmodified peptide is used in place of the candidate peptide in the split intein-based selection system described herein along with an inducible promoter operably linked to a negative selection marker and driving expression of the negative selection marker. The cells may be contacted with the negative selection agent to deplete cells with an unmodified peptide that binds to the target protein of interest. Without being bound by a particular theory, formation of a full-length transcription factor and subsequent expression of the full-length transcription factor would be dependent on the unmodified peptide binding to the target peptide in this system.
Expression of a reporter gene may be detected by any suitable method known in the art, including by analysis of RNA (e.g., reverse transcription-polymerase chain reaction (RT-PCR)), by analysis of protein levels (e.g., immunoassays), by analysis of enzyme activity (e.g., analysis of catalytic activity), by contacting cells with one or more selection agents, or by fluorescence analysis. A reporter protein may be detected by any known method, including via fluorescence microscopy, an immunoassay (including a western blot or an ELISA), or flow cytometry.
As one of ordinary skill in the art would appreciate, any transcriptional or translational output may be coupled with the first and second fusion proteins described herein.
In some embodiments, the intein-based selection system comprises a fusion protein with (i) a first fragment of a reporter protein, (ii) a first split intein, and (iii) a target protein, and another fusion protein that comprises (i) a candidate peptide, (ii) a second split intein, and (iii) a second fragment of the reporter protein. The first and second split inteins are complementary fragments, such that association of the first split intein with the second split intein promotes trans-splicing. In this embodiment, the presence of a full-length reporter protein is indicative of the candidate protein binding the target protein.
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of natural products that are modular and engineerable. In RiPP biosynthesis, the ribosome synthesizes a peptide using proteinogenic (i.e., amino acids that are biologically incorporated into proteins during translation) amino acids, and modifying enzymes subsequently bind to the peptide and modify it. Such post-translational modification introduces chemical diversity beyond the 20 standard amino acids, as well as structural diversity such as macrocyclization. Each modifying enzyme is constrained by a set of design rules, such as which amino acid(s) they can modify, the recognition site(s) (RSs) they will associate with, the distance (e.g., number of amino acids) between the RS and the amino acid residue(s) to be modified, and the amino acid context in which they can act (e.g., the amino acids in proximity to the target amino acid(s) that they modify). Synthetic peptides with particular activity (e.g., desired biological activity), and libraries thereof, can be constructed by incorporating the design constraints of one or a combination of modification enzymes into a peptide synthesis scheme.
In some embodiments, a RiPP comprises a leader amino acid sequence and a core amino acid sequence. In some embodiments, the leader and the core are connected via a cleavable linker (e.g., a protease-cleavable linker). In some embodiments, a RiPP comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more) recognition sites (RSs) for one or more distinct modification enzymes.
Aspects of the present disclosure relate to peptides for identification of binders to a target protein (e.g., candidate peptides or a plurality thereof) and peptides that may be useful in clinical applications. A candidate peptide is a peptide whose binding activity to a protein is being investigated. In some embodiments, a peptide comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 6-25 or 26-45, an amino acid sequence in Table 3 or any amino acid sequence disclosed herein, including fragments thereof.
The peptides described herein may be modified (e.g., the peptide may comprise a non-natural amino acid, a non-naturally occurring linkage, and/or a post-translational modification). In some embodiments, a modified peptide comprises a post-translational modification. In some embodiments, a modified peptide is produced recombinantly. In some embodiments, a modified peptide is produced synthetically. Without being bound by a particular theory, recombinant production of a modified peptide using a host cell may require expression of one or more protein modification enzymes. In some embodiments, the peptide is non-naturally occurring. In some embodiments, the peptide is naturally occurring.
Without being bound by a particular theory, a peptide comprising one or more modifications may be more stable (e.g., has reduced denaturation at a particular temperature), have increased bioavailability, and/or have increased solubility compared to a peptide not comprising the one or more modifications.
Non-limiting examples of post-translational modifications include formation of thioether bridges, formation of ester bridges, phosphorylation, glycosylation, acetylation, ubiquitylation/sumoylation, methylation, palmitoylation, myristoylation, prenylation, hydroxylation, GPI anchoring, ADP-ribosylation, pyrrolidone carboxylic acid, citrullination, S-nitrosylation, sulfation, amidation, nitration, oxidation, gamma-carboxyglutamic acid, topaquinone, lysine topaquinone, phosphopantetheine, quinone, hypusine, iodination, bromination, cysteine tryptophylquinone, formylation, and tryptophan tryptophylquinone.
In some embodiments, a peptide described herein is a ribosomally synthesized and post-translationally modified peptide (RiPP). RiPPs are ribosomally-produced peptides that comprise a post-translational modification. There are several subfamilies of RiPPs and RiPPs are grouped based on the biosynthetic machinery that produce the RiPP and structural characteristics. See, e.g., Table 1 below, which is based on Table 1 from Ortega and van der Donk, Cell Chem Biol. 2016 Jan. 21; 23(1):31-44; and Arnison et al., Nat Prod Rep. 2013 January;30(1):108-60.
In some embodiments, a modified peptide comprises two or more (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20) non-contiguous amino acids that are linked. In some embodiments, a modified peptide comprises at least 1 pair, at least 2 pairs, at least 3 pairs, at least 4 pairs, at least 5 pairs, at least 6 pairs, at least 7 pairs, a least 8 pairs, at least 9 pairs, at least 10 pairs, at least 15 pairs, at least 20 pairs, at least 30 pairs, at least 40 pairs, or at least 50 pairs) of non-contiguous amino acids that are linked. As a non-limiting example, scaffold L1 in
In some embodiments, a peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, a least 24, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 thioether bridges. In some embodiments, a peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, a least 24, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 ester bridges. In some embodiments, a peptide comprises a thioether bridge and an ester bridge.
As an example, lanthipeptides comprise Lan and/or MeLan thioether bis-amino acids. In some embodiments, a peptide is a lanthipeptide. In some embodiments, a lanthipeptide comprises scaffold Li: AACX1X2X3X4X5X6MPPX7X8X9X10X11X12C (SEQ ID NO: 1), wherein: X6 and X7 are each the amino acid S or T; X1-X5 and X8-X12 are each any amino acid; and the peptide comprises a thioether bridge that links C at position 3 to S or T at position 9 in SEQ ID NO: 1 and a thioether bridge that links S or T at position 13 to C at position 19 in SEQ ID NO: 1. See, e.g., L1 in
In some embodiments, a peptide is a microviridin. Microviridins may comprise lactones made from Glu/Asp and Ser/Thr side chains and/or lactams made from Lys and Glu/Asp residues. In some embodiments, a microviridin comprises X1PX2TTX3X4TX5X6X7EX8X9DX10DEX11X12X13 (SEQ ID NO: 2) (scaffold L2), wherein: X2 is the amino acid H, Q, N, K, D, or E; X6 is the amino acid F, L, S, I, M, T, V, or A; X7 is the amino acid F, L, I, or V; X1, X3-X5 and X8-X13 are each any amino acid; and the peptide comprises an ester bridge that links T at position 5 of SEQ ID NO: 2 to D at position 15 of SEQ ID NO: 2 and an ester bridge that links T at position 8 of SEQ ID NO: 2 to E at position 12 of SEQ ID NO: 2. See, e.g., L2 in
In some embodiments, a peptide comprises a sactipeptide (ranthipeptide). Sactipeptides comprise one or more intramolecular thioether linkages between Cys side chains and α-carbons of other amino acids. In some embodiments, a sactipeptide comprises: X1CX2X3X4X5X6CX7X8X9X10X11 (SEQ ID NO: 3) (scaffold L3), wherein: X5 and X10 are each the amino acid D or E; X1-X4, X6-X9, and X11 are each any amino acid; and the peptide comprises a thioether bridge that links C at position 2 to D or E at position 6 of SEQ ID NO: 3 and a thioether bridge that links C at position 8 to D or E at position 12 of SEQ ID NO: 3. See, e.g., L3 in
In some embodiments, a sactipeptide comprises X1CX2X3CX4X5X6X7X8X9 (SEQ ID NO: 4) (scaffold L4), wherein: X4 and X7 are each the amino acid D or E; X1-X3, X5-X6, and X8-X9 are each any amino acid; and the peptide comprises a thioether bridge that links C at position 2 to D or E at position 6 of SEQ ID NO: 4 and a thioether bridge that links C at position 5 to D or E at position 9 of SEQ ID NO: 4. See, e.g., L4 in
In some embodiments, a peptide described herein has biological activity, e.g., antimicrobial activity. In some embodiments, peptides having antimicrobial activity are modified from RiPPs of microbiome bacteria from a subject, such as a human subject. Non-limiting examples of bacteria from which RiPPs can be modified to have antimicrobial activity include the Flavobacteria, Proteobacteria, Actinobacteria, Erysipelotrichia, Clostridia, Bacilli provided in
In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34) consecutive amino acids of the sequence GSX1GX2X3GVX4X5TX6SHECHMNTWQFLX7TCCS (SEQ ID NO: 95), wherein: X1 is R or G; X2 is G, W, or K; X3 is D, Q, or N; X4 is M, L, or F; X5 is H, P, or K; X6 is L, V, or I; and X7 is L, F, or A;. In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from GGDGVMHTLTHECHMNTWQFLLTCC (SEQ ID NO: 90), GSRWWQGVLPTVSHECRMNSFQHIFTCC (SEQ ID NO: 92), and GGKNGVFKTISHECHLNTWAFLATCCS (SEQ ID NO: 93). In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) comprises a sequence selected from GGDGVMHTLTHECHMNTWQFLLTCC (SEQ ID NO: 90), GSRWWQGVLPTVSHECRMNSFQHIFTCC (SEQ ID NO: 92), and GGKNGVFKTISHECHLNTWAFLATCCS (SEQ ID NO: 93).
In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42) consecutive amino acids of the sequence GWX1WGSYRDX2YGALRGPNX3X4FVGX5GGX6X7X8X9X10X11X12X13X14SWRLVPR (SEQ ID NO: 102), wherein: X1 is I, F, L, or Y; X2 is V or I; X3 is P, S, T, or K; X4 is P, G, N, or R; X5 is L, G, A, or R; X6 is V, F, or S; X7 is P, T, or S; X8 is P, G, or E; X9 is G or W; X10 is G or R; X11 is V or L; X12 is S or V; X13 is G or P; and X14 is G or R. In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from GLIYGKYRDVLSGARLVTPPEVALRLVPR (SEQ ID NO: 103), GWFWGSYRDIFGALRGPNSGFEGGGGFTGGGVSGGSWRLVPR (SEQ ID NO: 104), GWLWGSYRDVYGVWHGPRTNFNGAGGSSEWRLVPR (SEQ ID NO: 105), and GWYWGNRRDIYGALRYANKRLVPR (SEQ ID NO: 106). In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) comprises a sequence selected from GLIYGKYRDVLSGARLVTPPEVALRLVPR (SEQ ID NO: 103), GWFWGSYRDIFGALRGPNSGFEGGGGFTGGGVSGGSWRLVPR (SEQ ID NO: 104), GWLWGSYRDVYGVWHGPRTNFNGAGGSSEWRLVPR (SEQ ID NO: 105), and GWYWGNRRDIYGALRYANKRLVPR (SEQ ID NO: 106).
In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GVGYbbYWGILPLVbKNPQIAPVaENbVKARLL (SEQ ID NO: 107), wherein ‘b’ is dehydrobutyrine and ‘a’ is dehydroalanine, and wherein a thioether bridge connects the dehydrobutyrine at position 15 to the alanine at position 21, and a thioether bridge connects the dehydrobutyrine at position 27 to the alanine at position 30. In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) comprises the sequence GVGYbbYWGILPLVbKNPQIAPVaENbVKARLL (SEQ ID NO: 107), wherein ‘b’ is dehydrobutyrine and ‘a’ is dehydroalanine, and wherein a thioether bridge connects the dehydrobutyrine at position 15 to the alanine at position 21, and a thioether bridge connects the dehydrobutyrine at position 27 to the alanine at position 30.
In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence provided in Table 12 (SEQ ID NOs: 115-147).
In some embodiments, a peptide having antimicrobial activity is selectively active against a particular class, genera, species, or strain of bacteria. In some embodiments, a peptide having antimicrobial activity does not kill commensal bacteria of a subject. In some embodiments, a peptide having antimicrobial activity kills pathogenic bacteria. In some embodiments, a peptide having antimicrobial activity is selective towards pathogenic bacteria over commensal bacteria. In some embodiments, a peptide having antimicrobial activity is selective towards bacteria of a first class, genera, species, or strain over bacteria of a second class, genera, species or strain. In some embodiments, being selective towards a first population of bacteria over a second population of bacteria means the peptide kills bacteria of the first population of bacteria at a concentration that is at least 5% lower (e.g., at least 10% lower, 15% lower, 20% lower, 25% lower, 30% lower, 35% lower, 40% lower, 45% lower, 50% lower, 55% lower, 60% lower, 65% lower, 70% lower, 75% lower 80% lower, 85% lower, 86% lower, 87% lower, 88% lower, 89% lower, 90% lower, 91% lower, 92% lower, 93% lower, 94% lower, 95% lower, 96% lower, 97% lower, 98% lower, or 99% lower) than the concentration that is required to kill bacteria of the second population. In some embodiments, being selective towards a first population of bacteria over a second population of bacteria means the peptide is capable of killing bacteria of the first population, but is unable to kill bacteria of the second population.
In some embodiments, a peptide having antimicrobial activity (e.g., a modified RiPP) disclosed herein comprises one or more post-translational modifications, such as modifications effected by one or more enzymes listed in Tables 5, 7, 8, 13, 14, and 17. Possible peptide post-translational modifications include, but are not limited to, phosphorylation (e.g., of serine, threonine, or tyrosine residues); glycosylation (e.g., N-glycosylation, O-glycosylation, glypiation, C-glycosylation, and phosphoglycosylation); ubiquitylation/ubiquitination; S-nitrosylation; methylation (e.g., N-methylation or O-methylation); N-acetylation; lipidation (e.g., C-terminal glycosyl phosphatidylinositol (GPI) anchor, N-terminal myristoylation, S-myristoylation, or S-prenylation); deamidation; eliminylation; prenylation; ADP-ribosylation; hydroxylation; polypeptide backbone modifications (e.g., stereoisomerization, dehydration, oxidation, cyclization), and any other post-translational modifications disclosed herein. Post-translational modifications are described further in Müller Biochemistry 2018, 57(2):177-187 (doi: 10.1021/acs.biochem.7b00861) and deGruyter et al. Biochemistry 2017, 56(30):3863-3873 (doi: 10.1021/acs.biochem.7b00536).
In some embodiments, one or more serine (S) and/or cysteine (C) residues of a peptide having antimicrobial activity disclosed herein is replaced with a dehydroalanine (e.g., by dehydration of a serine or cysteine). In some embodiments, one or more threonine (T) residues of a peptide having antimicrobial activity disclosed herein is replaced with a dehydrobutyrine (e.g., by dehydration of a threonine). In some embodiments, a peptide having antimicrobial activity (e.g. a modified RiPP) disclosed herein comprises one or more thioether bridges, one or more thioester bridges, and/or one or more other bridges. Any modified peptide disclosed herein can comprise any combination of post-translational modifications described herein (e.g., one or more dehydrated amino acids, one or more thioether bridges, one or more thioester bridges, and/or one or more other bridges).
Despite the structural diversity of RiPPs, RiPP biosynthesis generally begins with production of a precursor peptide by ribosomes; the precursor peptide generally comprises an N-terminal leader sequence and a C-terminal core sequence that comprises sites for post-translational modification. In some embodiments, biosynthesis requires a C-terminal recognition sequence. The leader sequence recruits the biosynthetic machinery and is, in some embodiments, cleaved by a peptidase to form a mature peptide. In some embodiments, a protein modification enzyme is a peptidase that cleaves the leader peptide.
In some embodiments, one or more protein modification enzymes (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) protein modification enzymes may be expressed in a cell to produce a modified peptide. In some embodiments, the protein modification enzyme is expressed from a heterologous nucleic acid. The expression of one or more protein modification enzymes may be under the control of an inducible promoter.
Protein modification enzymes including RiPP synthesis enzymes are known. As a non-limiting example, Prochlorosin (ProcM) is a member of the enzyme class that installs the macrocyclic thioether linkages that give rise to lanthipeptides. ProcM engages in dehydration-based chemistry that targets side chain serine/threonine residues. ProcA is a natural peptide substrate for ProcM. TgnB is a member of the enzyme class that installs the macrocyclic ester linkages that give rise to microviridins. TgnA is a natural peptide substrate for the modifying enzyme, TgnB. PapB is a member of the enzyme class that installs the macrocyclic thioether linkages that give rise to ranthipeptides, or sactipeptides. Freyrasin (PapB) engages in radical-based chemistry that targets main chain carbon atoms of aspartate/glutamate residues. LynD is a cyanobactin cyclodehydratase (PDB ID 4V1T). Additional non-limiting examples of protein modification enzymes including RiPP synthesis enzymes are provided in Table 7. See also, e.g., Ortega and van der Donk, Cell Chem Biol. 2016 Jan. 21; 23(1): 31-44. In some embodiments, a protein modification enzyme comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 80-83, 174, 176, 179, 180, 183, 185, 187, 188, 190, 192, 247, 249-251, 253, 255, 256, 258, 262, 264, 265, 267, 270, 271, 274, 275, 279-281, 285, 289, 290, 292, 295, 296, 298, 300-303, 305, 308-310, 312, 313, 316, 318-322, 325, 327, 330, 332, 334, 335, 337, 338, 342, 343, 346, 349, 350, 354, 356, 360, 362, and 363. In some embodiments, a protein modification enzyme comprises a sequence selected from SEQ ID NOs: 80-83, 174, 176, 179, 180, 183, 185, 187, 188, 190, 192, 247, 249-251, 253, 255, 256, 258, 262, 264, 265, 267, 270, 271, 274, 275, 279-281, 285, 289, 290, 292, 295, 296, 298, 300-303, 305, 308-310, 312, 313, 316, 318-322, 325, 327, 330, 332, 334, 335, 337, 338, 342, 343, 346, 349, 350, 354, 356, 360, 362, and 363. See, e.g., Table 4, Table 7, Table 8, Table 9, and Table 17.
In some embodiments, the split intein-based selection methods described herein comprise sequencing to identify the candidate peptide in the host cell. In some embodiments, a host cell comprises a plasmid encoding the candidate peptide and the plasmid may be sequenced. Non-limiting examples of sequencing methods include next-generation sequence (NGS), nanopore sequencing, and Sanger sequencing.
Provided herein are methods for engineering RiPPs, such as to develop non-naturally occurring RiPPs with desired properties. Both the leader and core sequences of a RiPP can be engineered based on the methods provided. In a leader sequence, recognition site(s) (RS) for protein modifying enzymes can be engineered (e.g., added, removed, optimized, or moved), such as to enable the use of the corresponding protein modifying enzyme to incorporate a particular post-translational modification to a peptide, or to prevent a particular protein modifying enzyme from acting on a given RiPP. In a core sequence, the amino acid sequence can be engineered, such as to facilitate post-translational modification by a particular protein modifying enzyme.
The amino acid sequence of a RiPP (including its leader and core sequences, as well as any additional amino acids within the RiPP) determine which protein modifying enzymes interact with the RiPP. Leader-dependent protein modifying enzymes associate with an RS within the leader sequence of a RiPP, and facilitate modification of an amino acid or amino acids within the core sequence. Tailoring protein modification enzymes associate with a particular amino acid or amino acids within the core sequence of a RiPP, and facilitate modification of one or more of those amino acids.
To engineer a RiPP, e.g., so as to include a particular set of post-translational modifications on a peptide having a particular amino acid sequence, the protein modification enzymes that facilitate the particular set of post-translational modifications are first identified. Consensus leader RS sequences for each leader-dependent enzyme are then compiled. Each leader RS sequence is then incorporated (e.g., by encoding in a nucleic acid sequence to be translated into the RiPP) into the leader sequence of the engineered RiPP. In embodiments in which one or all of the RS sequences for a given engineered RiPP have constraints on the distance between the RS and the amino acid(s) to be modified, each RS is placed in the leader sequence according to its respective constraint(s). An optimized leader sequence can be identified by screening candidate leaders and calculating a position score (e.g. by quantifying the amount of peptide having the desired modification pattern for each candidate leader sequence and identifying the leader sequence generating the highest yield of modified peptide). A non-limiting example of this screening process to identify optimized leader sequences is demonstrated in
The RiPP engineering method provided herein enables the synthesis of a given peptide comprising a particular amino acid sequence with a specific combination of post-translational modifications. Biosynthesis using engineered RiPPs, rather than chemical or other conventional synthesis mechanisms, has one or more benefits, including but not limited to increased yield, decreased cost, and decreased complexity of the synthesis relative to alternative synthesis methods (e.g., chemical synthesis).
To engineer a RiPP, it may also be desirable to build a library of RiPPs to be screened with a particular protein modification enzyme or a particular combination of protein modification enzymes to identify preferred RiPPs (e.g., having a particular desired property or combination of properties) that comprise the desired post-translational modifications. Degenerate peptide libraries (i.e., libraries in which each amino acid of each member of the library is chosen randomly from all 20 natural amino acid options) can be designed, but have the disadvantage of being too large to be screened by conventional means (or in some instances are too large to be synthesized). For example, a degenerate library of peptides of 8 amino acids in length comprises peptides with 2.56×1010 distinct amino acid sequences, a number which is impossible or unfeasible to synthesize and/or screen. Such libraries are either impossible or unfeasible to synthesize and/or screen based on cost (sequencing, materials/reagents, etc.), time, or other considerations. As such, provided herein are libraries of RiPPs comprising a plurality of peptide members defined by a particular amino acid sequence motif. A library of RiPPs, in some embodiments, comprises peptides that are each 5-100 amino acids (e.g., 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, or any range or combination thereof) in length. A library, in some embodiments, comprises peptides that are each defined by a particular amino acid motif X1X2X3X4 . . . Xn, wherein n is the number of amino acids within the peptide (i.e., the length of the peptide), wherein each of X1-Xn is independently chosen from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, and wherein at least one of X1-Xn (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of X1-Xn) is chosen from fewer than 20 amino acids. In some embodiments, at least one of X1-Xn is restricted to a single amino acid. As a non-limiting example, X1 may be chosen from 3 amino acids, X2 may be chosen from 7 amino acids, X3 may be chosen from 2 amino acids, and so on. In some embodiments, the amino acid motif X1X2X3X4 . . . Xn is determined to be susceptible to modification by 1, 2, 3, 4, 5, 6, 7, 8, or more distinct protein modification enzymes. In some embodiments, the plurality of peptides of the library do not have random amino acid sequences.
In some embodiments, a library comprises peptides defined by a particular amino acid motif determined to be susceptible to modification by 1, 2, 3, 4, 5, 6, 7, 8, or more distinct protein modification enzymes. In some embodiments, less than 100% (e.g., less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%) of the members of the peptide library are capable of being fully modified by the protein modification enzymes to which the amino acid motif was determined to be susceptible. In some embodiments, at least 1% (e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) of the members of the peptide library are capable of being fully modified by the protein modification enzymes to which the amino acid motif was determined to be susceptible.
In some embodiments, each member of a library disclosed herein comprises a SUMO tag. In some embodiments, each member of a library disclosed herein comprises a SUMO tag at its 5′ end. In some embodiments, each member of a library disclosed herein comprises a SUMO tag at its 3′ end. In some embodiments, each member of a library disclosed herein comprises a SUMO tag and a histidine tag at its 5′ end (e.g., the member comprises the structure [histidine tag]-[SUMO tag]-peptide or [SUMO tag]-[histidine tag]-peptide). In some embodiments, each member of a library disclosed herein comprises a SUMO tag and a histidine tag at its 3′ end (e.g., the member comprises the structure peptide-[histidine tag]-[SUMO tag] or peptide-[SUMO tag]-[histidine tag]-peptide). In some embodiments, each member of a library disclosed herein comprises a SUMO tag and a histidine tag at its 5′ end or at its 3′ end. In some embodiments, a histidine tag is a hexahistidine tag. In some embodiments, each member of a library disclosed herein comprises a tobacco etch virus protease (TEVp) cleavage site, or each member comprises two TEVp cleavage sites. In some embodiments, each member of a library disclosed herein comprises a TEVp cleavage site in between a RiPP peptide and a SUMO tag (e.g., the member comprises the structure peptide-[TEVp site]-[SUMO tag] or [SUMO tag]-[TEVp site]-peptide).
In some embodiments, a plurality of host cells comprises a library of peptides disclosed herein. In some embodiments, each host cell comprises a peptide of the library (e.g., each host cell comprises a peptide of the library and the peptide comprised by each host cell is independent of the peptides comprised by each other host cell). In some embodiments, each host cell is a bacterial cell. In some embodiments, each host cell comprises a nucleic acid sequence encoding the peptide. In some embodiments, each host cell further comprises a protein modifying enzyme. In some embodiments, the protein modifying enzyme is encoded by a nucleic acid sequence comprised by the host cell.
In some embodiments, a library is synthesized in a plurality of host cells. For example, in some embodiments, each member of the library is synthesized in a separate host cell. In some embodiments, each host cell is a bacterial cell. In some embodiments, a library is synthesized in a population of bacteria. In some embodiments, each bacterium of the population expresses a single member of the library. In some embodiments, each member of the library is synthesized in a host cell in which one or more protein modifying enzymes are also expressed.
In some embodiments, a library is capable of being screened by methods disclosed herein (e.g., using split-intein based selection). In some embodiments, screening of a library disclosed herein identifies one or more peptides with a desired functional property (e.g., a desired biological property). In some embodiments, screening of a library disclosed herein identifies one or more peptides with antimicrobial activity. In some embodiments, screening of a library disclosed herein identifies one or more peptides with binding activity to a target protein.
The target protein may be any protein of interest. In some embodiments, a target protein is a cell surface receptor, antigen, transmembrane protein, glycoprotein, glycolipid or any other cell surface macromolecule. In some embodiments, the target protein is a viral protein or a fragment thereof. In some embodiments, the target protein comprises a receptor binding domain (RBD) from a coronavirus protein. In some embodiments, the coronavirus is 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome, or MERS), SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS), or SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or COVID-19). In some embodiments, the target protein is a bacterial protein or a fragment thereof. In some embodiments, the target protein is a bacterial enzyme. In some embodiments, the target protein is a bacterial outer-membrane protein. In some embodiments, the target protein is a bacterial toxin. In some embodiments, the target protein is a bacterial structural protein. In some embodiments, the target protein is a bacterial polymerase. In some embodiments, the target protein is a bacterial transcription regulator.
In some embodiments, the target protein is SARS-CoV-2 receptor binding domain (RBD) of the Spike protein. Spike protein is a surface glycoprotein that binds to angiotensin I converting enzyme 2 (ACE2) to promote viral entry. The al helix of ACE2 makes most of the binding contacts with the RBD and is provided as SEQ ID NO: 72.
In some embodiments, the target protein comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 71 (RBD). In some embodiments, the target protein comprises the amino acid sequence of SEQ ID NO: 71.
In some embodiments, the target protein comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 72 (al helix of ACE2). In some embodiments, the target protein comprises the amino acid sequence of SEQ ID NO: 72.
Non-limiting examples of known cellular receptors include ACVR2A, EGFR/HER1, HER2/ERBB2, ERBB3/HER3, CD32a/FCGR2A/Fc gamma RIIa, CD32b/FCGR2B/Fc gamma RIIb, CD16a/Fc gamma RIIIa, CD16b/Fc gamma RIII, CD155/PVR, TNFR1/TNFRSF1A/CD120a, TNFR2/TNFRSF1B/CD120b, 4-1BB/TNFRSF9/CD137, TRAIL R2/CD262/TNFRSF10B, TRAIL R4/CD264/TNFRSF10D, TNFRSF11A, TRAIL R1/CD261/TNFRSF10A, TRAILR3/TNFRSF10C, TACI/TNFRSF13B(CD267) HVEM/TNFRSF14/CD270, BCMA/TNFRSF17/CD269, GITR/TNFRSF18/CD357, FGFR2/CD332, CD23/FCER2, FCRL1/FCRH1, TIM-3/HAVCR2, IL1RL1/IL-1 R4, IL17RA/IL-17RA/CD217, IL-4R/CD124, IL7R/IL-7R/CD127, TrkA/NTRK1, PDGFRB/CD140b, TREM-2/TREM2, ACVR2B/Activin RIIB, FCGRT & B2M, CD89/FCAR, IL3RA/CD123, IGF1R/CD221/IGF-I R, Insulin Receptor/INSR/CD220, LILRB2/ILT4/LIR-2, VEGFR2/KDR/Flk-1/CD309, MCSF Receptor/CSF1R/CD115, EPHA3/Eph Receptor A3, CD16-2/FCGR4, FcERI/FCER1A, TIM-1/KIM-1/HACVR, IL6R/IL-6R/CD126, LILRB4/CD85k/ILT3, IL2RA/IL-2RA/CD25, CD122/IL-2RB, LDLR/LDL R/LDL Receptor, CD112/Nectin-2/PVRL2, and TFRC/CD71.
A peptide described herein may have a particular binding affinity for a target protein. Binding affinity is the apparent association constant or KA. The KA is the reciprocal of the dissociation constant (KD). The peptides identified by the methods described herein may have a binding affinity (KD) of at least 10−5, 10−6, 10−7, 10−8, 10−9, 10−10 M, or lower for a target protein. An increased binding affinity corresponds to a decreased KD. Higher affinity binding of a peptide for a first protein relative to a second protein can be indicated by a higher KA (or a smaller numerical value KD) for binding the first protein than the KA (or numerical value KD) for binding the second protein. In such cases, the peptide has specificity for the first protein (e.g., a first protein in a first conformation or mimic thereof) relative to the second protein (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). In some embodiments, the peptides described herein have a higher binding affinity (a higher KA or smaller KD) to an appropriate protein as compared to the binding affinity of the same type of peptide produced using naturally occurring secretion signal peptides. Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 105 fold. In some embodiments, any of the peptides produced as provided herein may be further affinity matured to increase the binding affinity of the peptide to the target protein or epitope thereof.
Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Non-limiting exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation:
[Bound]=[Free]/(Kd+[Free])
It is not always necessary to make an exact determination of KA, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA, FACS analysis or magnetic immunoprecipitation, which is proportional to KA, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.
In some embodiments, a peptide disclosed herein or identified through the methods disclosed herein decreases the binding affinity of a target peptide with a naturally occurring cognate binding partner. In some embodiments, a peptide disclosed herein or identified through the methods disclosed herein decreases the binding affinity of a target peptide with a naturally occurring cognate binding partner by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.
Aspects of the present disclosure provide host cells comprising any of the nucleic acids, fusion proteins, peptides, enzymes, selection markers and components of the split intein-based systems disclosed herein. In some embodiments, a host cell is a eukaryotic cell. In some embodiments, a host cell is a prokaryotic cell. In some embodiments, a host cell is a bacterial cell. In some embodiments, a host cell is an E. coli cell. As one of ordinary skill in the art would appreciate, components of the split intein-based systems disclosed herein may be selected based on the type of host cell used.
A nucleic acid may encode any of the fusion proteins, peptides, enzymes, selection markers and components of the split intein-based systems disclosed herein. As used herein, a heterologous nucleic acid is one that is introduced into a host cell. A nucleic acid, generally, is at least two nucleotides covalently linked together, and in some instances, may contain phosphodiester bonds (e.g., a phosphodiester “backbone”). A nucleic acid is considered “engineered” if it does not occur in nature. Examples of engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids.
Nucleic acids encoding any of the fusion proteins, peptides, enzymes, selection markers and components of the split intein-based system described herein may be introduced into a host cell using any known methods, including but not limited to chemical transfection, viral transduction and electroporation. In some embodiments, one or more nucleic acids that are introduced into a host cell integrate into the host cell genome; in some embodiments, one or more nucleic acids that are introduced in a host cell do not integrate into the host cell genome. The nucleic acids described herein may encode one or more of the fusion proteins, peptides, enzymes, selection markers and components of the split intein-based system disclosed herein. In some embodiments, a nucleic acid comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 47-66 or 73-79, a nucleic acid sequence in Table 3, or a nucleic acid sequence disclosed herein. In some embodiments, a nucleic acid comprises a nucleotide sequence of any one of SEQ ID NOs: 47-66 or 73-79, a nucleic acid sequence in Table 3, or a nucleic acid sequence disclosed herein. Any of the plasmids disclosed herein may be used.
It should be understood the methods of identifying peptides disclosed herein may or may not use host cells. In some embodiments, a split intein-based system disclosed herein is not used in a host cell. For example, in vitro methods comprising incubating a split intein-based system disclosed herein in a reaction vessel under suitable conditions is encompassed by the present disclosure.
Any of the host cells, nucleic acids, fusion proteins, peptides, enzymes, selection markers and components of the split intein-based systems disclosed herein, in some embodiments, may be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. A kit may include one or more containers housing the components of the disclosure and instructions for use. Specifically, such kits may include one or more agents described herein, along with instructions describing the intended application and the proper use of these agents. In certain embodiments, agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents. Kits for research purposes may contain the components in appropriate concentrations or quantities for running various experiments.
In some embodiments, the instant disclosure relates to a kit for identifying a peptide that binds a target protein, the kit comprising a container housing any of the host cells, nucleic acids, fusion proteins, peptides, enzymes, and components of the split intein-based systems disclosed herein. In some embodiments, the kit further comprises instructions for identifying the peptide and/or performing the split intein-based selection.
In some embodiments, the instant disclosure relates to a kit comprising a container housing any of the nucleic acids disclosed herein. In some embodiments, the kit comprises a container housing a nucleic acid that comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 47-66 or 73-79, a nucleic acid sequence in Table 3, or a nucleic acid sequence disclosed herein; or that comprises the nucleotide sequence of any one of SEQ ID NOs: 47-66 or 73-79, a nucleic acid sequence in Table 3, or a nucleic acid sequence disclosed herein. In some embodiments, the instant disclosure relates to a kit comprising a container housing any of the peptides disclosed herein. In some embodiments, the kit comprises a container housing a peptide that comprises a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 6-25 or 26-45, an amino acid sequence in Table 3 or any amino acid sequence disclosed herein, including fragments thereof; or that comprises the amino acid sequence of any one of SEQ ID NOs: 6-25 or 26-45, an amino acid sequence in Table 3 or any amino acid sequence disclosed herein, including fragments thereof. In addition, kits of the disclosure may include instructions, a negative and/or positive control, containers, diluents and buffers for the sample, sample preparation tubes and a printed or electronic table of reference peptide sequences for sequence comparisons.
The kit may be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable (e.g., reconstitutable) or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration. The kit may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject. The kit may include a container housing agents described herein. The agents may be in the form of a liquid, gel or solid (powder). The agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively, it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely. Alternatively, the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more or all of the components required to administer the agents to an animal, such as a syringe, topical application devices, or IV needle tubing and bag, particularly in the case of the kits for producing specific somatic animal models.
The kit may have a variety of forms, such as a blister pouch, a shrink-wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kit may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kit may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.
Any of the peptides (e.g., modified peptides) disclosed herein or identified by a method disclosed herein may be formulated in a pharmaceutical composition for administration to a subject. As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In all embodiments, human subjects are preferred.
In some embodiments, the subject is a suspected of having a disease or has previously been diagnosed as having a disease. In some embodiments, the subject is a human suspected of having a disease, or a human having been previously diagnosed as having a disease. Methods for identifying subjects suspected of having a disease may include physical examination, subject's family medical history, subject's medical history, biopsy, viral tests (e.g., nasal swabs), antibody tests (e.g., serological testing), or a number of imaging technologies such as ultrasonography, X-ray imaging, computed tomography, magnetic resonance imaging, magnetic resonance spectroscopy, or positron emission tomography.
In some embodiments, the subject is suspected of having or has previously been diagnosed as having an infectious disease (e.g., a disease caused by a pathogen and/or virus). As a non-limiting example, the subject may have coronavirus disease 2019 (COVID-19), which is an infectious disease. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 may be diagnosed using any suitable method including nasopharyngeal swabs and serology testing for antibodies against coronavirus.
In some embodiments, the subject is suspected of having or has previously been diagnosed as having cancer. The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstram's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).
In some embodiments, the subject is suspected of having or has previously been diagnosed as having a bacterial infection (e.g., an infection caused by a pathogenic bacterium). Exemplary bacterial infections include, but are not limited to, pulmonary infections (e.g., upper respiratory infection or lower respiratory infections), urinary tract infections, skin infections (e.g., bacterial cellulitis), sexually transmitted infections, neurological infections (e.g., bacterial encephalitis, bacterial meningitis), cardiac infections (e.g., bacterial endocarditis, bacterial myocarditis, or bacterial pericarditis), gastrointestinal infections (e.g., gastric infections, bacterial gastroenteritis, bacterial pharyngitis), bacterial vaginosis, and Lyme disease. Bacterial infections can be caused by any bacterium, including, but not limited to, Gram-positive bacteria, Gram-negative bacteria, Streptococcus pneumoniae, Haemophilus species, Staphylococcus aureus, Mycobacterium tuberculosis, methicillin-resistant S. aureus, non-typhoidal Salmonella species, Salmonella typhi, Bacillus cereus, Clostridium perfringens, Clostridium botulinum, Escherichia coli (ETEC, EPEC, EHEC, EAEC, EIEC), Salmonella sp., Shigella sp., Campylobacter sp., Yersinia enterocolitica, Clostridium difficile, Vibrio cholerae, Vibrio parahemolyticus, Listeria monocytogenes, Aeromonas hydrophila, Plesiomonas sp., Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Borrelia burgdorferi, Vibrio cholerae, Clostridium tetani, and Bacillus anthracis.
A “plurality” of elements, as used throughout the application refers to two or more of the elements.
The peptides (e.g., modified peptides) of the invention are administered to the subject in an effective amount for detecting or modulating protein (e.g., enzyme) activity. An “effective amount”, for instance, is an amount required to confer therapeutic effect on a subject, either alone or in combination with at least one other active agent. The effective amount of a peptide of the invention described herein may vary depending upon the specific peptide used, the mode of delivery of the peptide, and whether it is used alone or in combination. The effective amount for any particular application can also vary depending on such factors as the disease being assessed or treated, the particular peptide being administered, the size of the subject, or the severity of the disease or condition as well as the detection method. One of ordinary skill in the art can empirically determine the effective amount of a particular molecule of the invention without necessitating undue experimentation. Combined with the teachings provided herein, by choosing among the various active peptides and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective regimen can be planned.
Pharmaceutical compositions of the present invention comprise an effective amount of one or more agents, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
General considerations in the formulation and/or manufacture of pharmaceutical agents, such as compositions comprising any of the engineered cells disclosed herein, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
Suitable routes of administration include, for example, parenteral routes such as intravenous, intrathecal, parenchymal, or intraventricular injection.
A three plasmid system was used to conduct selection experiments. All plasmids are low-medium copy number variants previously characterized1: the “peptide plasmid” is a pSC101 backbone with an ampicillin resistance cassette (working concentration of 100 ng/uL) and contains a Type IIs restriction site for insertion of RiPP/peptide sequences N-terminal to one half of the split intein/sigma factor under control of an inducible T5 lacO promoter (maximally induced with 1 mM IPTG). The “modifying enzyme plasmid” is a p15A backbone with a spectinomycin resistance cassette (working concentration of 50 ng/uL) and contains a Type IIs restriction site for inserting cognate RiPP modifying enzymes under control of an inducible pCym promoter (maximally induced with 100 uM cumate). The “selection plasmid” is a ColE1 backbone with a kanamycin resistance cassette (working concentration of 50 ng/uL) and contains two regions of expression. The first is a C-terminal fusion of the SARS-CoV-2 receptor binding domain (RBD) of the Spike protein2 to the other half of the split intein-sigma factor. The second expression region contains two open reading frames downstream of the ECF20_992 promoter. The first is a sfGFP-cat gene for expression of superfolder-green fluorescent protein (sfGFP) and a chloramphenicol acetyltransferase (CAT) and the second is hsvTK-mScarlet-I gene for expression of the red fluorescent protein mScarlet-I and, when in the presence of a nucleoside analog, the toxic gene product, herpes simplex virus thymidine kinase (HsvTK) 3 (
The three plasmid system allows for flexible selection methods. Inducible expression of the peptide and modifying enzyme plasmids results in production of modified RiPP libraries with C-terminal fusions to the split intein machinery. RiPPs that are able to bind to the target (in this case, the RBD) lead to productive intein association and splicing 4 of the split sigma factor, which induces expression of the selection cassettes. For positive selection of binders, increasing concentrations of chloramphenicol (cm) can be used to enrich for target binders (in this case, an RBD-intein fusion) that produce increasing amounts of CAT (
For the generation of this initial round of RBD hits, a negative selection was not implemented. Current and future selections will utilize positive and negative selections in consecutive, discrete rounds to best evolve RiPP libraries toward high affinity and specific binders to the RBD.
Five libraries were designed based on in-house understanding of RiPP biosynthetic constraints, (
Library sizes were as indicated in Table 2 based on serial dilutions and counting colony forming units (CFU)/mL.
Appropriate antibiotics were used at every stage for plasmid propagation, as detailed above. Inducers were used at maximum concentration where indicated, as detailed above. Transformation efficiencies were recorded via serial dilution and CFU/mL counts. Libraries were miniprepped and transformed into separate electrocompetent strains of E. coli Marionette-Clo5 containing cognate modifying enzyme and selection constructs (transformation efficiencies >108 CFU/mL). After a one-hour outgrowth, strains were diluted 1:50 for plasmid outgrowth and induction of library peptides and modifying enzymes. This culture was grown overnight at 30° C., with shaking at 250 RPM.
After overnight growth, libraries were diluted 1 mL in 100 mL TB medium in inducing conditions. Selections were grown at 30° C. for 20 hours, 250 RPM. 4 mL of each selection was miniprepped and modifying enzyme/selection plasmids were restriction digested using SacI/KpnI (NEB, per manufacturer's instructions). Resulting digests were column purified (Zymo) and re-transformed in strains containing modifying enzyme/selection plasmids. This step was done in order to eliminate escape mutants in the selection plasmid (for instance, mutations generating high-level, constitutive expression of cat-GFP; see
For this initial pilot screen, 3 rounds of positive selections were conducted, at 300, 800 and 1200 uM chloramphenicol. Cell populations were assessed via cytometry to observe shifts in REU values (
20 sequences were codon optimized, synthesized as gBlocks (IDT), and individually cloned into the peptide plasmid. These 20 peptide plasmids were co-transformed with the PapB modifying enzyme plasmid and either the RBD-intein or Mdm2-intein as target in the selection plasmid. After overnight induction of peptide/modifying enzyme at 30° C., cells were analyzed via cytometry and REU values determined (
Chemically-modified peptides are made by all kingdoms of life, where the enzymatic decorating and reshaping are critical for function. Peptides could be designed de novo by harnessing the modifying enzymes from the deluge of genomics, but it is difficult to extract the rules guiding their use and combination. In this Example, a model that captures the minimal specificity constraints was developed to use enzymes gleaned from microbial gene clusters encoding RiPPs (ribosomally-synthesized and post-translationally modified peptides). They include the recognition site (RS) sequence and restrictions on its placement in the precursor peptide and the tolerance to variability of the released core. The rule sets were empirically parameterized using a pipeline to construct and evaluate the activities of enzymes against hundreds of precursor peptide variants in Escherichia coli. This was applied to nine enzymes from eight RiPPs classes, including those for which there is little prior characterization (lactone macrocyclase, tyramine excisionase, glutamate heterocyclase, cysteine heterocyclase, glycosyltransferase, serine kinases, decarboxylase, and methyl transferase). The rules can be algorithmically combined to computationally design new-to-nature RiPPs, demonstrated by creating a 13-mer that combines excision, heterocyclization, and phosphorylation (PlpXY, LynD, ThcoK). Formalizing enzyme rules provides a foundation for retrosynthesis, where peptides and libraries could be designed to facilitate therapeutic discovery and diversification.
Across biology, peptides are chemically modified for diverse purposes, from enhancing antimicrobial potency to honing signaling specificity and nucleating inorganic materials [1-6]. In the pursuit of pharmaceutical or other applications, one would like to design patterns of modifications in a peptide, but this is challenging using total synthesis because routes are long and involve highly-functionalized and chiral molecules [7-9]. An alternative would be to encode the peptide as a gene that is expressed with enzymes that introduce the desired post-translational modifications (PTMs) [10-12]. The process of identifying a path to a target molecule is a form of retrosynthesis that requires knowing the rules by which enzymes can be combined to act on a peptide sequence [13].
Peptide secondary metabolites are often encoded in genomes as a RiPP where a precursor peptide is expressed that comprises a leader and core sequence [3]. An enzyme binds to a recognition site (RS) in the leader and modifies amino acid(s) in the core [14-16]. PTMs include the introduction of cycles, added moieties (e.g, methylation), or conversions (e.g., epimerization) [3, 17-19]. A leader can have up to three RSs, sometimes overlapping to save space [20-22]. Changing the distance dbetween the RS and the modified amino acid(s) can affect the efficiency and which amino acids are modified [22-31]. Some enzymes are more sensitive than others, likely due to flexibility or allostery [32-34]. Leader-independent “tailoring” enzymes add modifications before or after the proteolytic release of the core [3]. To date, up to eight modifying enzymes have been found to act on a single peptide (theiostrepton), but the number of modifications can be much larger (e.g., polytheonamide has 49 modifications by 7 enzymes) [22, 35, 36].
During evolution, core hypervariability around a PTM scaffold facilitates the exploration of functional space, for example to diversify antimicrobials against new threats [10, 11, 37-40]. By physically separating binding from catalysis, leader-dependent enzymes are highly tolerant to changes to the core sequence; typically, 40-90% of mutants are modified correctly [12, 16, 17, 19, 20, 26, 27, 31, 41-50]. The specificity of tailoring enzymes can vary, with some being sensitive to sequence or the peptide conformation and others being very broad, notably when they modify the termini [46, 51-55]. Taken together, the minimal rule set needed to repurpose an enzyme is: 1. the tolerance of the core sequence, and 2. the RS sequence and position constraints within the leader, if relevant (
Various approaches have been used to discern these rules. Importantly, when characterizing an enzyme for retrosynthesis, the constraints must be with respect to the chemistry performed and not function [42]. For example, in one study, only 41% of thiopeptide mutations that yielded the correct PTM also retained antibiotic activity [56]. While bioinformatics can be used to deduce the RS or enumerate core variability, drawing them from natural genomes implies functionality [57, 58]. Another approach is to evaluate the impact of mutants with libraries created though alanine-scanning, saturation mutagenesis, or core shuffling [42, 47, 59-66]. Billions can be evaluated using assays that screen for function or by panning for target binding [26, 47, 56, 62, 64, 67-72]. The throughput of chemical assays is more limited; electrospray ionization mass spectroscopy (ESI-MS) can characterize hundreds of variants [29, 33, 42, 73]. MALDI-MS and SAMDI-MS could scale to 104 variants or more, but they are currently limited by peptide length and require additional expensive processing steps when automated [31, 50, 56, 74, 75].
Early work has combined enzymes from different pathways to build novel compounds, but typically, these have been sourced from the same RiPPs family [39, 55, 76]. Some tailoring enzymes will modify nearly any core and this observation has been used to incorporate methyltransferases, decarboxylation or epimerases into unrelated pathways [46, 55, 77]. Combining enzymes across RiPP classes has proven more difficult. In pioneering work, Mitchell and van der Donk showed that leader-dependent enzymes from sactipeptide, lanthipeptide, and heterocycloanthracin pathways could be combined by creating leader chimeras combining the RSs [74]. Along with a tailoring enzyme, this was used to make a new 32-mer lanthipeptide containing a thiazoline and d-Alanine.
In this Example, enzyme specificity rules were formalized to facilitate their algorithmic combination to create a peptide with a defined PTM pattern. Four leader-dependent enzymes (TgnB, PlpXY, PaaP and LynD) and five tailoring enzymes (PalS, ThcoK, PadeK, EpiD, LasF) were selected to represent diverse chemical modifications, species, and RiPP classes (Table 5) [18, 54, 59, 78-81]. Most have little prior information in the literature regarding substrate preferences. Escherichia coli was selected as the chassis because RiPP enzymes often work in this host and the “Marionette” strains allow the independent control of up to a dozen genes [82-84]. An N-terminal SUMO RiPP stabilization tag (RST; as described in Example 8) was used to increase the concentration of precursor peptide and simplify leader cleavage, which can be difficult to predict [85]. Mutagenesis strategies were developed to efficiently extract the enzyme rules: recognition site, distance constraint, and core tolerance (
A microtiter-based peptide expression, purification, and analysis pipeline was adapted to study modification of many peptide mutants/variants by individual modifying enzymes. This is a two plasmid system, with modifying enzyme produced from a p15A medium-copy plasmid and precursor peptide expressed from a pSC101 origin mutated to maintain at medium copy number (var 2, [87],
Bacillus thuringiensis
Pantoea agglomerans
Pleurocapsa sp.
Prochloron spp.
Lentzea kentuckyensis
Aeribacillus pallidus
Staphylococcus epidermidis
Thermobacillus composti
Paenibacillus dendritiformis
Nine RiPP modifying enzymes were selected for analysis in this Example (Table 5; see also Table 7). Four of the selected enzymes were leader dependent and needed recognition sites and spacing constraints elucidated. Three of those (PlpXY, LynD, and PaaA) contained the RiPP recognition element (RRE) domain previously shown to be responsible for leader binding [14, 15], while TgnB is an ATP-Grasp microviridin-class enzyme with a less-studied binding mechanism. These four enzymes are from different bacteria genera, catalyze diverse chemical modifications, and result in different physicochemical properties in the modified peptide:
(1) TgnB, from Bacillus thuringiensis, covalently links glutamate/aspartate residues with serine/threonine residues to form the bi-cyclic depsipeptide thuringeinin[58]. The resulting cyclic peptide is a potent antidigestive (digestive protease inhibitor) and is rigid and constrained, both properties of interest in the peptide drug-discovery community [6]. The enzyme was codon optimized and synthesized, and used to modify a truncated peptide substrate with only one core (versus the three-core repeat in the native TgnA peptide)[58].
(2) PlpXY, from Pleurocapsa sp. PCC 7319, excises tyramine (the amine, alpha carbon, and sidechain of tyrosine) by breaking the peptide backbone and re-fusing it, resulting in a ketone containing beta-amino acid [18]. The modification is interesting both in its chemical reactivity (it can be used as a click substrate), and its uniqueness—no other RiPP enzyme known alters the peptide backbone as extensively. The enzyme PlpX and its RiPP recognition element PlpY were both codon-optimized and expressed as a two-gene operon and used to modify PlpA2, one of three core peptides in the cluster.
(3) PaaA, an antibiotic from Pantoea agglomerans, performs a Claisen condensation between two adjacent glutamate residues, resulting in the fused-ring heterocycle indolizidine [78]. This alkaloid moiety is not typically associated with RiPP biosynthesis, but is prevalent in many bioactive small molecules [91]. The enzyme was codon optimized and was used to modify its native precursor peptide (also codon optimized).
(4) LynD, from Lyngbya sp., dehydrates a cysteine with a peptide backbone amide to form a five-membered heterocycle. The resulting heterocycle, thiazoline, spans what was the amide bond, creating a protease resistant backbone[92]. Thiazolines retain the planar structure of the amide [92] and can be oxidized to aromatic thiazoles by cyclodehydratases found in some RiPP clusters[93]. Due to their valuable properties, thiazol(in)e heterocycles are frequently found in bioactive natural products and approved drugs[92]. LynD was codon optimized, and was used to modify a single-core truncation of TruE, a precursor peptide from a homologous pathway.
To generate the peptide expression plasmids, and leader mutants thereof, some were ordered as oligos, PCR amplified, and cloned into TypeIIs expression vectors, but a majority were synthesized and assembled by Twist Biosciences. From Twist, peptide vectors were rehydrated and immediately co-transformed with their cognate modifying enzyme plasmid in microtiter 96-well plates. Because only clonal, sequence verified plasmids were used, co-transformants were directly selected for by growing in LB supplemented with kanamycin and carbenicillin, without plating on agar and picking colonies. After overnight incubation, stationary phase cultures were diluted 1:100 into expression media and maximally-induced at approximately mid-log to decrease potential toxicity effects on growth[94]. A high-velocity microtiter plate shaker was required due to the use of deep 96-well plates. It was found that shaking below 900 r.p.m. led to cell sedimentation and highly variable expression. The peptide/enzyme expressions were conducted in TB media, such that conditions for all enzymes were identical.
Liquid-chromatography coupled to mass spectrometry (LC-MS) was used for peptide analysis. SUMO-tagged peptides were analyzed directly (without tag removal) in order to decrease the number of processing steps and reduce peptide-to-peptide run variability (the tag buffers against the chromatographic properties and solubility of diverse peptides). Peptides purified and eluted via IMAC were directly injected on the LC-MS for analysis. Since all of the modifications studied in this Example resulted in a change in mass between the unmodified and modified peptide, extracted compound chromatograms could be generated based on the expected masses of the unmodified, partially modified (if relevant), and modified peptides. If a chromatogram contained a peak, it was fit with a skewed gaussian[96], and the resulting fit was used to calculate peak area. Peak areas for modified, partially modified (if applicable), and unmodified peptide were summed to calculate the total peptide observed, which was then used to calculate the fraction of each peptide modification state.
While this process was chosen due to its simplicity and scalability, it does have two limitations: 1) Modified, partially modified, and unmodified peptide masses were sometimes not fully resolved in the MS. For the tagged large peptides analyzed (15-25 kDa), the isotope distribution could span 15-25 Da. If the modification being studied caused a mass shift of <15-25 Da, the isotope distributions between the unmodified and modified peptides would not be fully resolved, leading to crossover during integration of the modified and unmodified peptides. Similarly, spurious sodium adducts could cause a 22 Da mass shift, resulting in overlap with enzyme-catalyzed 14 Da (LasF) and 18 Da (TgnB and LynD) mass shifts, also affecting integrations and fraction modified calculations. 2) Multiple charge states are required to reliably annotate a peptide as present, which raises the limit of detection. On the machine that was used, the SUMO-tagged peptide limit of detection was estimated to be a peak area of ˜104-5. The median peak size observed was ˜106, meaning that a peptide with a fraction modified of 0.0 could actually have been as high as 0.1, if the modified peptide intensity was just below the detection threshold, or fraction modified of 1.0 could have been as low as 0.9 (though this would have had no effect on intermediate values of fraction modified). Most of the overlapping isotope effects were solved by extracting ECCs using a small m/z window around the expected mass of each peptide, such that regions of isotope overlap were ignored. For any remaining effects of overlapped isotope distribution, as well as sodium adduct and high limit of detection effects, the effects should largely have been dependent on the modification mass shift and the peptide being studied. Therefore, the effects could be countered by only comparing fraction modified within the same modification, since the effects should be similar (and cancel out) for similar peptides with the same modification.
Using the outlined pipeline, the four leader-dependent modifying enzymes were used to assay for modification (
Identification of Recognition Sites within Leaders
A simple approach was taken to deduce each enzyme's RS sequence(s). Alanine scanning is effective in finding the RSs, by measuring when the modification to the core is disrupted [60]. However, making a single substitution at every position is inefficient, particularly for long leaders and provides unnecessary resolution given that the smallest RS known is 7 amino acids[98] (excluding protease sites). Instead, blocks of 4-5 alanines were used to scan the leader and measure the impact on the fraction modified (block size dependent on leader length). The block was iteratively moved by 2-3 residues for each mutant (
A thermodynamic model was derived to infer the per-residue contribution to the binding of the modifying enzyme. This was simplified by assuming that the reaction follows Michaelis-Menten kinetics, where reversible binding to the leader precedes modification and release. This treats the binding and unbinding as being at quasi-steady state with respect to the production and degradation of the peptide; in other words, the ratio modified ρ, is the equilibrium value. Then, the change in the free energy of binding of the variant n with respect to the wild-type is
where R is the gas constant and T is temperature. If the contribution of each residue i of a mutant contributes additively to the free energy change, then
ΔΔGn=Σi=1MΔΔGi (Equation 2)
where M is the number of mutated residues. An algorithm was developed to assign ΔΔGi values using all of the variant data. Initially, the contribution of ΔΔGn was divided equally amongst the mutated residues (for example, divided by 5 for a 5-alanine block in which none of the wild-type residues replaced by the block were originally alanines). However, some residues were mutated in two variants, so the residue was assigned a ΔΔGi value of the mean of the two ΔΔGn/M values. The resulting ΔΔGi assignments violated equation 2 (ΔΔGi values will not sum to ΔΔGn within a variant), so ΔΔGi values were adjusted iteratively and in small increments (similar to a force-directed graph) until the constraint of equation 2 was satisfied for all variants.
The result of this calculation is shown in
One source of additional information was leader structure. A Deep Convolutional Neural Field algorithm (RaptorX Structure Property Prediction) was used to predict the secondary structure of the leaders (
Sequence conservation within peptide homologs was also incorporated. Encouragingly, for all of the leader peptides, regions of high ΔΔGi values corresponded to regions of high conservation in weblogos of peptide homologs (
While the alanine scans showed that sequences in the RSs are necessary, and homologous sequences and structural predictions can help validate those data and inform boundaries, they did not prove that the RS is sufficient for modification. For each of the peptides, truncations were tested to remove sequence that should be unnecessary. The TgnA RS is at the N-terminus of the leader, so only truncations between the RS and the core were possible. The effect of truncations on RS-to-modification spacing versus sequence importance could not be differentiated, but truncations of various sizes were generally tolerated. Most truncations were modified over half as well as wild-type, and were modified as well as or better than similarly-sized insertions, indicating that the modifying enzyme is sensitive to changes in RS-modification site spacing. Previously reported deletions scanned through the TgnA leader also agreed with annotation of the TgnB RS as necessary and sufficient for modification, where only deletions that included RS residues were unmodified [58]. Both the TruE and PlpA2 peptides included sequences N-terminal to the RS, removal of which was well-tolerated by each respective modifying enzyme, with fraction modified similar to that of full-length leader (
The final recognition site sequences are outlined in boxes in
Variants were designed to alter the spacing d between the RS and the modified residue. An alternative would be to define d as the distance to the start of the core sequence, which could be more intuitive for enzymes that modify multiple core amino acids, such as TgnB [58]. However, the distance to the modification was selected as it was more likely to be the physical distance to the modification site itself that influences modification rather than the distance to the core/leader cleavage site. Additionally, during forward engineering of precursor peptides, it functions as a constraint on core length by keeping modifications from being allowed at infinite core positions away from the leader. As such, d was defined as the number of residues between the RS and the modified amino acid. If multiple amino acids were modified (for example the two lactone cycles in TgnB modification), it was the distance to the first modified amino acid.
Changing d from its optimal value was expected to lead to lower modification efficiencies. In its simplest form, this can be treated as an energy well, where a wider well corresponds to more core positions being modifiable if RS position in the leader is kept constant. In contrast, a steep well indicates that the modification can only occur at a single residue, optimally spaced from the RS. A spring model is the simplest way to model this effect, which has been applied to similar biophysical phenomena, such as modeling the impact on ribosome binding that results from different spacing between the Shine-Delgarno and ATG start sites [88]. Using a spring model, RS-to-modification distances less than optimal would be “stretched” for modification, while distances greater than optimal would be “compressed”. The following equation can be derived from Hooke's Law,
where d0 is the optimum spacing, κs and κc are the stretching and compression spring constants, and H(x) is a step function. Equation 3 could be changed to reflect other functions; for example, it might take on the form of a steep step function if there is a distance at which suddenly an enzyme is no longer active. It also does not have to be monotonic, with more complex forms modeling enzymes that exhibit multiple local minima or periodic behavior. In its current form, the stretching and compression constants define the width of the energy well described above, with small values of κ corresponding to a wide energy well with high spacing tolerance and large values corresponding to a narrow energy well with low spacing tolerance.
Leader variants were designed for each modifying enzyme to perturb the RS spacing, starting with TgnB. TgnA* has 35 residues between the RS and the first modified residue, with 31 of those being in the leader. Five truncation variants were designed by removing residues at the C-terminus of the leader, starting with two amino acids and increasing in increments of four amino acids to the longest truncation of 18 amino acids, representing over half of the spacer. Three insertion variants were also designed using a TEV cleavage site (amino acid sequence ENLYFQ (SEQ ID NO: 111)) and glycines as a spacer: the TEV site alone is a 6 amino acid insertion, TEV site followed by triple-glycine is +9 amino acids, and TEV site flanked by triple-glycines is +12. Each of these 8 variants was assayed for modification, and the fraction modified for the variants is shown in
aParameters for Equation 3.
bNo indel tolerated; Fit for ΔΔGn = 20 at d-d0 = 1
PlpXY is known to be tolerant to varying core positions, since there are two precursor peptides associated with the cluster that have RS to modification distances of 6 (PlpA2) and 21 (PlpA1). The leader peptide (and RS sequence) of PlpA1 differs from PlpA2, so modification of the two was not directly compared, since modification differences due to distance cannot be separated from RS sequence differences. Instead, spacing parameters were elucidated similarly to TgnA*, using engineered insertion/deletion variants of PlpA2. Since the RS is one residue away from the C-terminus of the leader peptide and the modified tyrosine is also close to the N-terminus of the core, only three deletion variants were tested: deletion of the final glycine (−1), the final glycine and first two residues of the core (−3), and the final glycine and first four residues of the core (−5). The same insertion variants were tested as for TgnA*/B: insertion of a TEV cleavage site (+6), TEV cleavage site followed by a triple-glycine (+9), and TEV cleavage site flanked by triple-glycines (+12). The variants were assayed for modification, with variant effect on modification converted to ΔΔGn and fit with spring constants (
The PaaA RS has very rigid placement restrictions (
LynD, and homologous cyanobactin heterocyclases, are known to be tolerant to spacing changes in the precursor peptide [39, 42]. In nature, it modifies the LynE peptide, which includes the same “LAELSEEAL (SEQ ID NO: 110)” RS defined in the truncated TruE* peptide, with three tandem cores and modified cystines spaced 9, 12, 24, 27, 39, and 42 amino acids from the RS [39]. In the full-length TruE peptide, which was modified with LynD in this Example, LynD modifies cysteines in two tandem cores, with RS-to-modification distances of 6 and 27 amino acids (
Libraries varying the core of each RiPP were made to determine modifying enzyme tolerance to different amino acids. In general, the approach of using scanning site saturation mutagenesis (SSSM) was followed and applied to positions surrounding the modified residue(s) [56, 59]. Degenerate oligonucleotides, with codons replaced by NNK mixed bases, were used to build libraries and isolate core sequence variants. Typically, a single residue would be varied at a time, with all single-residue NNK libraries pooled together such that an individual library member has a random amino acid at a single random position (also known as a saturation mutagenesis single variant library or single codon randomization library, abbreviated as sSSSM for single SSSM). The pooled oligonucleotide libraries were cloned and individual variants were isolated and sequence verified. To increase coverage at each position, the number of core positions in the libraries was decreased and included only those surrounding and necessary for the modification. For cores with long C-terminal “tails” after the modification, truncations were made to the peptide's C-terminus to determine the minimal sequence necessary for modification. All four modifications were close to the N-terminus of the core, so the entire core N-terminal to the modification was always included in the libraries. PaaA and TgnB modifications used wild-type leaders for modification, while leaders with long N-terminal regions before the RS (TruE* and PlpA2) used N-terminal leader truncations shown to be sufficient for modification during leader/RS characterization (
The raw data for the TgnA* core library are shown in
Although the TgnA* library was designed to generate single-mutant variants, several variants were isolated with two mutations and one with three, which provided an opportunity to investigate mutation additivity (
For PlpA2 modification by PlpXY, truncations to the C-terminus were first investigated to identify residues necessary for modification. Increments of three amino acids were removed from the C-terminus of the peptide until modification broke. Removal of 12 amino acids was tolerated, with fraction modified within error of modification of the wild-type peptide, while removal of 15 amino acids was not modified at all. This was in agreement with previous work which showed that the proline at position 11 was necessary for modification [18]. Based on this data, a library was built to include positions 1-12 of the core peptide. A similar sSSSM library was built as described for TgnA*, with 41 single-mutation variants isolated and assayed. In contrast to TgnA*, only half of the variants were tolerated, with one variant removed because of high variance amongst replicates (
Based both on the six cysteines modified by LynD in the native LynE substrate [39] and the two cysteines modified in the TruE substrate, LynD was anticipated to be extremely permissive of different amino acid residues surrounding the modified cysteine residue. In the TruE* peptide, both the entire core (five amino acids preceding the modification) and the follower (four amino acids after the modification) were included in the library, with the follower treated as core peptide rather than a structural element (similarly to PaaP follower in its library). Given the number of residues in the library, and the potentially high tolerance of diverse amino acids, a saturation mutagenesis library of all positions simultaneously was used, allowing the core sequence to be xxxxxCxxxx (SEQ ID NO: 112), where x is any amino acid. A single degenerate oligonucleotide, with all core and follower codons except the cysteine replaced by NNK, was used to build the library. In the resultant variants, peptides with more than one cysteine were screened out, since it was impossible to tell which ones were modified via LC-MS. Twenty-four variants were isolated and assayed, in addition to 10 variants that were synthesized to have charged and/or bulky polar residues flanking the modified cysteine (native flanking residues are usually small and/or hydrophobic). All of the custom/designed variants were well modified, showing that LynD tolerated charged or bulky polar side chains at the modification site. Of the 24 random variants, 17 were modified above the half-of-wild-type threshold. At all of the positions included in the library, tolerated amino acids were physiochemically diverse, consistently including 5-6 of the 6 physicochemical groupings used to classify amino acids (positive, negative, polar, aliphatic, aromatic, G/P). Based on this, the motif was trimmed to include only the positions adjacent to the modified cysteine. Those two positions were updated to allow 19 amino acids, all except cysteine, since modification of adjacent cysteines was not investigated (
The same expression/analysis pipeline described for leader-dependent modifying enzymes was applied to leader-independent tailoring enzymes. Tailoring enzymes do not bind recognition sites in the leader, instead they bind directly to the site of modification in the core, with specificity presumably determined by the amino acids around the modification. As such, these enzymes have no RS or RS spacing constraints, but do have core sequence constraints that can be elucidated similarly to the core constraints of leader-dependent modifying enzymes. To maintain consistency between all enzymes, expression conditions were equivalent to those described for modifying enzymes: peptides were expressed as a SUMO fusion and expressed and modified in TB media in 96-well plate format.
Of the nine enzymes selected for characterization (Table 5), five were leader-independent tailoring enzymes. One of the enzymes modifies the side chain of an internal peptide residue while others modify the C-terminal residue side chain or carboxyl group. In contrast to the leader-dependent modifying enzymes, where all were from different RiPP classes, three of the five tailoring enzymes came from lasso peptide clusters, highlighting the compatibility of lasso peptide tailoring enzymes have with heterologous expression in this platform. The tailoring enzymes catalyze diverse transformations and have been sourced from diverse bacterial species (Table 5):
(1) EpiD is an oxidative decarboxylase from the epidermin biosynthetic pathway, a type 1 lanthipeptide antibiotic identified from Staphylococcus epidermidis[105, 106]. It is an integral tailoring enzyme for formation of the aviCys macrocyclization, though without the other enzymes in the pathway the aviCys cycle is not formed and decarboxylation results in an enethiolate [107], with a corresponding loss of mass of −46 Da. This modification is valuable both for its potential for forming constrained aviCys macrocycles[6] when combined with other enzymes and also for removing the carboxy group, decreasing polarity and potentially increasing membrane permeability[108, 109].
(2) PalS is a glycosyltransferase that catalyzes the class-defining glycosylation of pallidocin, a glycocin antibiotic[110]. In pallidocin, a cysteine is glycosylated, causing a gain of mass of +162 Da. Glycosylation can play diverse roles in small molecules, often used in antibiotics to inhibit peptidoglycan biosynthesis by glycopeptides[111] and now proposed as a strategy for improving peptide bioavailability during drug design[112].
(3) LasF is a methyltransferase from the lasso peptide antibiotic lassomycin[l13]. It methylates the carboxyl group on the C-terminus to form a methyl ester, causing a gain in mass of +14 Da. Similar to EpiD decarboxylation, the methyl ester is uncharged (unlike the carboxyl group), potentially aiding membrane permeability[108, 109].
(4) ThcoK and (5) PadeK are both kinases from lasso peptide clusters that install 1-3 phosphates on the C-terminal serine of their respective peptides[80, 81]. Because multiple phosphate groups can be added, the gain in mass can be +80, +160, and +240, corresponding to +1, +2, and +3 phosphates, respectively. Naturally, their biological role is unknown, but synthetically they can be used to modify substrate pKa/log P properties or create phosphopeptide mimetics that act as signal transduction inhibitors[114]. Both ThcoK and PadeK were included to enable phosphorylation of a greater number of peptides by investigating two kinases with presumably different sequence constraints. Since these enzymes install a variable number of phosphates, any number of phosphates to be “modified” was considered, meaning that fraction modified is the fraction of peptide that has 1, 2, or 3 phosphates installed.
Each of these tailoring enzymes catalyze a mass shift that can be assayed via LC-MS, in the same manner that leader-dependent modification was assayed. The five tailoring enzymes and their respective wild-type precursor peptides were first assayed for modification (
Similar to leader-dependent modifying enzymes, core motifs were elucidated using scanning site saturation mutagenesis. Since tailoring enzymes do not require the leader (or a majority of the core), most of the precursor peptide was truncated to investigate only those residues surrounding the modification. Each peptide library was limited to eight varying positions. For tailoring enzymes that modified the amino acid side chain (PadeK, ThcoK, and PalS), the modified residue was not included in the library since it was necessary for modification, so the total peptide size was truncated to 9 amino acids. For the two enzymes that modified the carboxy group on the C-terminus (LasF and EpiD), the C-terminal residue was included in the library, so the total peptide size was truncated to the C-terminal 8 amino acids. The positions were numbered based on their position in the wild-type (full-length) core, not their position in the truncated version.
Initial libraries varying single amino acids at a time (like those used with TgnB, PlpXY, and PaaA) resulted in variants that were well modified (
Finally, each motif was analyzed and minimized based on tolerated amino acids at each position. If every observed mutant at a position was accepted in the tolerance summary, and those tolerated amino acids spanned 4+ of the 6 physicochemical amino acid classes used, the position was annotated as unconstrained and allowed to be any amino acid. Unconstrained positions on the edge of a motif could then be removed from the motif entirely. During golden-gate/typeIIs assembly of the libraries, assembly bias that lowered the number of amino acid variants at the N- and C-termini of the library was observed, so terminal positions were often removed from the motif if they didn't meet the 4+ criteria above, but had unconstrained positions between them and the modified residue. For example, in the PadeK tolerance summary (
The EpiA peptide was truncated to include the eight C-terminal residues (positions 15 through 22). EpiD modification was investigated using sSSSM, dSSSM, and dfSSSM libraries, each of which were cloned separately and a total of 33 variants isolated and assayed between the libraries. For many of the variants, the replicates varied more than what was observed for other enzyme peptide variants. Analysis of the raw chromatograms showed large peaks that were above the detection limit, but the spectra were noisier than spectra from other peptides/enzymes, for unknown reasons. Despite the lower quality data, trends were visible: mutations close to the N-terminus were observed to be well modified and those close to the C-terminus (modification site) were poorly modified. Position 20 did not tolerate negatively charged aspartate/glutamate amino acids, while hydrophobic (L), polar (S, N, and Y), and positively charged (R) amino acids were tolerated. Positions 17, 18, and 19 were found to be very permissive and all mutations at positions 15 and 16 were tolerated, so positions 15-19 were removed from the core motif, which is shown in
The PalA peptide was truncated to include the four amino acids to either side of the glycosylated cysteine (9 amino acids total). Three libraries were designed: sSSSM, dSSSM, and dfSSSM, with 74 total variants assayed for modification by PalS (Supplementary Note 10). A majority of variants (40) were 100% modified, with only 14 variants showing intermediate levels of modification and the remaining 20 not tolerated. Of those that weren't tolerated, all but two included mutations flanking the modified cysteine (positions 24 and 26). The remaining two were G22F and G27I single mutation variants, both surprising given the diverse amino acids tolerated at both of those positions. While there were multiple examples of variants with overlapping amino acids at a position, investigating non-additivity was impossible, since most variants were not at quasi-steady state but were fully modified. Mutation S29G had a lower fraction modified (0.81) than S29G with Y28F (1.0), but was within the S29G standard deviation of +/−0.24. In another example, F23K was fully modified while F23K with G24R as poorly modified (0.19). Assuming additivity, G24R was the offending mutation, except F23G with G24R was well modified (0.79). This may be an example of non-additivity, but because the F23K single mutant variant was fully modified it's possible that the F23K mutation was detrimental to modification, but not enough to lower the fraction modified below 1.0. Only when combined with another slightly detrimental mutation, G24R, did F23K mutation bring modification down significantly. Without a clear indication of non-additivity, the core tolerance summary was assembled using all the variants, observed positions 21, 23, and 28 to be unconstrained, and updated the core motif to include positions 22-27 (
The LasA peptide was truncated to include the C-terminal eight amino acids, all of which were varied in the library. Both sSSSM and dSSSM libraries were constructed, with 37 variants isolated and assayed for modification. Mutations to LasF had greater impact on the activity of the enzyme compared to variants for other tailoring enzymes. None of the variants with multiple mutations were well modified, and only 5 single-mutant variants had wild-type levels of modification. Hydrophobic amino acids (A, V, L, F, and W) were generally allowed in all positions. Mutation of the C-terminal isoleucine to tyrosine and cysteine was not tolerated, in agreement with data for a LasF homolog showing mutation of the C-terminal residue led to a 4-fold reduction in methylation. The variant data was used to build the core tolerance summary (
PadeK and ThcoK were both truncated to include the C-terminal nine amino acids, with the final serine not included in the library since its side chain is modified. Both of these enzymes were very tolerant to diverse core sequences, so sSSSM, dSSSM, dfSSSM, and tSSSM libraries were all used to elucidate core constraints. In total, 31 PadeA variants and 34 ThcoA variants were tested. ThcoK was the most tolerant enzyme investigated: only one variant was below the modification threshold, with the mutation adjacent to the modified cysteine. Positions 16 through 21 all passed the criteria for being unconstrained, so positions 15 through 21 were removed from the motif, leaving only the modified serine, and the preceding residue. PadeK was more constrained: it only showed high specificity at the penultimate core residue and at core positions 22 and 21, respectively (adjacent to the ultimate/modified serine) (
Design of Peptides with Multiple PTMs
A design algorithm was developed to create a library of core variants enriched for a desired modification pattern (
Leader design proceeds by moving the RS sequences with respect to the core and calculating their contribution to a scoring function. The maximum leader length is a parameter that can be set in the algorithm, with a default value of L=40 amino acids. The score S of RS placement m is the predicted effect of RS-to-modification distance d compared to optimal distance d0.
which is bounded to the range 0-1 (inclusive). The total score for a RS placement in a leader p for a set of M enzymes is defined as
S
p=Πm=1NSm (Equation 5)
The algorithm then seeks to identify the optimum p that maximizes the score. This can be found simply by enumerating all possible placement combinations of the RS sequences.
There are several use cases in which it is beneficial to save space by overlapping the RS sequences, as sometimes occurs in natural leaders. For instance, the constraints on d might be too rigid to separate them. It could also free other space in the leader for additional enzymes to bind. Finally, shorter leaders could facilitate the use of specific DNA oligosynthesis techniques in building a library. To this end, an algorithmic approach was developed to evaluate overlapping RS sequences. If two RS sequences could overlap without any amino acid mismatches, then this was done without penalty. However, in most cases, overlap would require an imperfect RS for at least one enzyme. To capture this, an additional term was calculated to modify the score,
In Equation 6, a and b are the lengths of RS1 and RS2 and z is the number of mismatched residues (BLOSUM62 score less than or equal to 0) in the overlap of the two recognition sites. The fraction was bounded to the range of 0-1 (inclusive) and simply included in the product of terms for the total score (Equation 5). If more than two RSs were being combined, more than one pair of RSs may overlap, and Smn was calculated for each overlapping pair and included with Equation 5. At mismatched overlapping RS positions, a random choice between the two possible amino acids can be made, or one RS can be given priority over the other in selecting the amino acid.
Typically, if tolerated, a TEV protease site was included between the leader and the core so the core could be released and recovered after purification. When used, the TEV sequence constraints were treated as an additional leader-dependent modifying enzyme. The six amino acid TEV sequence ENLYFQ (SEQ ID NO: 111) was added as an RS, with fixed placement (high κ constants), such that it contributed to the calculation of Sp. TEV cleavage occurs after this sequence and was permissive to different amino acids at the first position of the core, except P, and reduced efficiency for L/E/I/V [115]. This core constraint was added as a core motif, with placement specified at position 1 of the core. In addition to this, there may be a gap between the RS sequences or between the last RS and the core. There are multiple options for filling these gaps provided by the algorithm: (1) GGS repeats; (2) choosing random amino acids (additional sequence constraints can be optionally added at any leader position); (3) spacer sequences taken from wild-type leaders of the enzymes being combined; and (4) nothing, the leader is returned with gaps to be filled in manually.
The final step was to design the core (
The algorithm was applied to design precursor peptides that can be modified by four enzymes: two leader-dependent modifying enzymes (LynD and PlpXY), one tailoring enzyme (ThcoK), and TEV protease (
A core motif was then designed by combining the rules associated with the three enzymes and including the restriction from the TEV protease that a proline cannot appear in the first position. Considering the variability allowed at each position, this resulted in 21,000 peptides that conformed to the rules. This was in contrast to the ˜1013 peptides that would result from all 20 amino acids being allowed at all non-modified positions. An oligo pool was built and designed to access a subset of the allowed peptides and cloned and sequence verified one that matched the enzyme restrictions and ten that had imperfect matches (
Expression and peptide modification was investigated in the same manner as for individual enzymes. Each of the eleven peptide plasmids were co-transformed with the multi-enzyme plasmid. Overnight cultures were diluted 1:100 into TB media, fully induced after 3 hours at 30° C., and incubated for 20 hours. Cultures were then lysed, affinity purified, and assayed via LC-MS.
All possible combinations of modification were searched (dehydration from LynD modification (−18 Da), tyramine excision from PlpXY modification (−135 Da), and phosphorylation from ThcoK modification (+80 Da)). For four of the peptides, masses were identified that matched expected triple-modification masses, suggesting a success rate of 80% for the hybrid core motif. The peptide variant with the highest fraction of triply modified peptide was selected for validation.
The co-transformed strain was struck out, and three colonies were individually grown up at small scale, affinity purified, and TEV cleaved. The final molecule was assayed via LC-MS/MS, where the mass and observed fragments matched the expected peptide structure.
This Example abstracted the substrate preferences of RiPP enzymes as “rules,” applicable to the constraint-based design of precursor peptides. Computational design can be used to guide the selection of enzymes to decorate a natural product [116], identify scaffolds to splice in a binding sequence [61, 117], or design large screening libraries enriched in modified peptides [62]. While RiPPs are generally very tolerant, the success rate declines rapidly as more constraints are added. For the example in
Chemical retrosynthetic planning algorithms use “rules,” extracted from the literature, to represent how a chemical moiety will be converted by a reaction [13, 119-121]. There is a trade-off between accuracy and path discovery: if every rule is specific to only one chemical, this would be the most reliable, but it would not be possible to predict paths to new chemicals. Algorithms balance these needs by specifying rules with respect to the number of atoms from the reaction center n; if n=0, then it is just the reaction itself and as n gets larger, this increases the accuracy as more of the chemical context is incorporated into the rule. This approach has been extended to enzymes using the same rules-based method of defining allowable enzyme substrates based on the substrate reaction center and surrounding atoms/functional groups [13].
Considering rules for RiPP enzymes, simply defining the chemistry performed by an enzyme and assuming perfect promiscuity for the other core positions is the philosophical equivalent to n=0. This assumption has implicitly appeared in the literature for RiPP design when highly tolerant enzymes were combined without restricting the core sequence [11, 23-25, 27]. Simultaneously, other retrosynthesis studies have engineered multiply modified peptides by generating peptide chimeras, with an enzyme effectively modifying its wild-type substrate [74, 76, 77], the equivalent of a large and un-engineerable n-value. The rules defined in this Example are the next level of constraints, representing the minimal information to capture substrate specificity. However, they incorporate a number of assumptions, including the additive combination of amino acid tolerances derived from single-mutant data. Indeed, incidences of non-additive compensatory effects from multiple mutations were observed. The next level of accuracy in rules could account for higher-order effects requiring more sequence knowledge of the core, such as charge, hydrophobicity, secondary structure, and loop entropy, all of which have been cited as important in determining RiPP enzyme specificity [22, 26, 42, 45, 47, 76, 122]. Similarly, in the leader it was assumed that recognition sites and spacing alone were determining factors of modification, but TgnB recognition site spacing variants varied in modification based solely on spacer sequence, indicating that leader sequence outside of the recognition site may affect modification (
However, many RiPP enzyme have properties, or gaps in knowledge, that make their function difficult to capture as a “rule.” Enzymes with wide RS spacing tolerance are often progressive, with difficult-to-predict behavior where single leader mutations change the modification pattern [20, 32, 34]. Kinetics are also a complicating factor, as enzymes in the same pathway can have orders-of-magnitude differences in time scales, from less than an hour to days[10, 33, 36, 124]. Imperfect leader sequences have been observed to alter enzyme kinetics, not just binding[33]. The order of operations also matters for cases in which later modifications require earlier ones to occur, for example, when a cyclization or epimerization orients an amino acid such that it is accessible for a subsequent modification[20, 30, 32, 61, 125, 126]. Tailoring enzymes can require that the released core peptide adopt a particular shape [42, 47, 52, 127].
This Example provides a new type of RiPP enzyme mining effort that differs from the approach of discovering new bioactive compounds by finding and reconstructing entire gene clusters from metagenomics data [65, 128]. Screens can be established to identify modifying enzymes along with simple approaches to define the minimal rule sets for their use. Because the goal is to combine them into a pathway, these enzymes need to be screened under a common set of conditions, whether it be in vivo or in vitro [76] and jettisoning those that do not work in this standardized context or that exhibit odd or unpredictable behaviors. These conditions may not reveal the precise role of enzymes in nature, but they provide the necessary information for forward design of artificial pathways. The “ideal” enzyme for retrosynthesis can also begin to be defined. One might think that it is a very tolerant enzyme regarding spacing to the modification, but broad substrate specificity can lead to unpredictable modification of multiple core residues and slow kinetics [33]. Instead, when given the option, it may better to have multiple enzymes on hand that differ in the distance from the RS where they modify their residue, such as appears to be the case in bottromycin biosynthesis [21]. Enzyme engineering, such as directed evolution, could be used to widen or tune substrate specificity specifically for the purpose of retrosynthesis. On last count, there are 300,000 RiPP clusters in the genomic databases with 4.6 million enzymes spanning ˜40 classes [129-134]. Finding subsets that work well together and characterizing their rules under common conditions would enable an enormous functional space to be algorithmically or combinatorially explored, providing unprecedented access to an emerging therapeutic modality: medium-sized constrained molecules, which are already showing promise for disrupting protein-protein interactions and other therapeutic targets that have traditionally been considered “undruggable”.
E. coli NEB 10-beta (C3019I, New England BioLabs, Ipswich, Mass., USA) was used for all routine cloning. E. coli NEB Express (C2523I, New England BioLabs, Ipswich, Mass., USA) was used to express precursor peptides with single modifying enzymes, and the Marionette derivative of E. coli NEB Express (Marionette X) was used to express precursor peptides with multiple modifying enzymes. Plasmids for precursor peptide expression and modifying enzyme expression were used as follows: precursor peptide genes used a pSC101 origin variant (var 2) [87] and single modifying enzyme plasmids contained p15A origins of replication and kanamycin resistance. Plasmids with multiple modifying enzymes contained p15A origins of replication and spectinomycin resistance. LB-Miller (B244620, BD, Franklin Lakes, N.J., USA) and TB (T0311, Teknova, Hollister, Calif., USA) supplemented with 0.4% glycerol (BDH1172-4LP, VWR, OH, USA) were used for peptide expression and modification. 2xYT liquid media (B244020, BD, Franklin Lakes, N.J., USA) and 2xYT+2% agar (B214010, BD, Franklin Lakes, N.J., USA) plates were used for routine cloning and strain maintenance. SOB liquid media (S0210, Teknova, Hollister, Calif., USA) was used for making competent cells. SOC liquid media (B9020S, New England BioLabs, Iwsich, Mass., USA) was used for outgrowth. Cells were induced with the following chemicals: cuminic acid ≥98% purity from Millipore Sigma (268402, Millipore Sigma, Saint Louis, Mo., USA) added as 1000× stock (200 mM) in EtOH or DMSO; isopropyl β-D-1-thiogalactopyranoside (IPTG) ≥99% purity (I2481C, Gold Biotechnology, Saint Louis, Mo., USA) added as 1000× stock (1 M) in water or DMSO. Cells were selected with the following antibiotics: kanamycin (K-120-10, Gold Biotechnology, Saint Louis, Mo., USA) as 1000× stock (50 mg/ml in H2O); carbenicillin (C-103-5, Gold Biotechnology, Saint Louis, Mo., USA) added as 1000× stock (100 mg/ml in H2O); spectinomycin (22189-32-8, Gold Biotechnology, Saint Louis, Mo., USA). Liquid chromatography was performed with Optima Acetonitrile (A996-4, Thermo Fisher Scientific, MA, USA) and water (Milli-Q Advantage A10, Millipore Sigma, Saint Louis, Mo., USA) supplemented with LC-MS Grade Formic Acid (85178, Thermo Fisher Scientific). The following solvents/chemicals were also used: Ethanol (V1001, Decon Labs, King of Prussia, Pa., USA), Methanol (3016-16, Avantor, Center Valley, Pa., USA), dimethyl sulfoxide (DMSO) (32434, Alfa Aesar, Ward Hill, Mass., USA), Imidazole (IX0005, Millipore Sigma, Saint Louis, Mo., USA), sodium chloride (X190, VWR, OH, USA), sodium phosphate monobasic monohydrate (20233, USB Corporation, Cleveland, Ohio, USA), sodium phosphate dibasic anhydrous (204855000, Acros, N.J., USA), guanidine hydrochloride (50950, Millipore Sigma, Saint Louis, Mo., USA), tris (75825, Affymetrix, Cleveland, Ohio, USA), TCEP (51805-45-9, Gold Biotechnology, Saint Louis, Mo., USA), and EDTA (0.5M stock, 15694, USB Corporation, Cleveland, Ohio, USA). DNA oligos and oligo pools were ordered from Integrated DNA Technologies (San Francisco, Calif., USA) and enzymes and peptide plasmids were assembled/cloned in-house or synthesized by Twist Biosciences (San Francisco, Calif., USA). Enzymes and peptides were codon optimized using an in-house optimization tool.
Saturated cultures in LB were diluted 1:100 into 1 ml TB in deep well plates, incubated for 3 hours (Multitron Pro, 30° C., 900 r.p.m.), supplemented with appropriate inducers, and incubated for an additional 20 hours (Multitron Pro, 30° C., 900 r.p.m.). For purification, plates were centrifuged (Legend XFR, 4,500 g, 4° C., 20 min), pellets were resuspended in 850 μl lysis buffer (5 M guanidinium hydrochloride, 300 mM NaCl, 50 mM sodium phosphate, pH 7.5), frozen (liquid nitrogen, −196° C.), thawed (Multitron Pro at 37° C., 900 r.p.m), and clarified via centrifugation (Legend XFR, 4,500 g, 4° C., 40 min). Peptides were affinity purified using His MultiTrap TALON plates (29-0005-96, GE Life Sciences), following manufacturer instructions, using 1×500 μl water and 2×500 μl lysis buffer for column equilibration, 2×500 μl wash buffer (300 mM NaCl, 50 mM sodium phosphate, 5 mM imidazole, pH 7.5), and 1×200 μl elution buffer (300 mM NaCl, 50 mM sodium phosphate, 150 mM imidazole, pH 7.5).
All chromatography was performed using mobile phases ACN (acetonitrile supplemented with 0.1% formic acid and 0.1% water) and water (supplemented with 0.1% formic acid). LC-MS was performed on one of two mass spectrometers: “QQQ” is an Agilent 1260 Infinity liquid chromatograph with binary pump configured in low-dwell volume mode, high-performance autosampler chilled to 18° C., and column oven, coupled to an Agilent 6420 QQQ mass spectrometer equipped with an Agilent electrospray ionization (ESI) source; nitrogen gas is supplied by a Parker Nitroflowlab and ESI source parameters are 350° C. gas temp at 12 L/min flow rate, 15 psi nebulizer voltage, 4000 V capillary voltage, 135 V fragmentor voltage, and 7 V cell accelerator voltage. “QTOF” is an Agilent 1260 Infinity II liquid chromatograph with binary pump configured in low-dwell volume mode and column oven set to 40° C., coupled to an Agilent 6545 QTOF mass spectrometer equipped with an Agilent electrospray ionization (ESI) source; nitrogen gas is building supplied and ESI source parameters are 350° C. gas temperature, 12 L/min gas flow, 30 psig nebulizer pressure, 350° C. sheath gas temperature, 8 L/min sheath gas flow, 3000 V capillary voltage, 1000 V nozzle voltage, 135 V fragmentor voltage, 15 V skimmer voltage, 600 V Oct 1 RF Vpp; the mass spectrometer was run in MS mode with reference mass enabled and tuned in positive mode with standard mass range (3200 m/z) and 2 GHz extended dynamic range.
mzXML files were parsed and imported into python to a long-form pandas dataframe and filtered for signals between 1-6 min and 500-2,500 Da. For each extract, the expected molecular weight of unmodified, modified, and partially modified (if applicable) peptides were calculated. For each molecular weight, all charge state [M+xH]x+ (x is number of protons/charges) masses were calculated and extracted as an EIC with a mass window of +/−5/x Da for extracts analyzed with “QQQ” and 2/x Da for extracts analyzed with “QTOF”. Charge state EIC intensities were summed together at each timepoint to generate an extracted compound chromatogram (ECC). If present, an ECC peak is fit with a skewed gaussian with parameters peak area, retention time, peak width, and peak skew. Peaks are considered real/trustworthy based on the following criteria: greater than 8 charge states present/observed at the same retention time (+/−0.2 min) with at least 4 being consecutive charge states, only one “large” peak in the ECC (i.e. no peaks greater than 80% of the largest peak height in the chromatogram), and not more than 2 “small” peaks (i.e. <3 peaks greater than 40% of the largest peak height), peak skew between 0 and 1.5, peak width less than or equal to 0.25. Within an extract, “total peptide” is defined as the sum of the peak areas of unmodified, modified, and partially modified (if applicable) peptides if the modification mass shift is >15 Da and is defined as the sum of the peak areas of unmodified and modified peptides otherwise (due to overlapping isotope distributions). Fraction modified is defined as the modified peptide peak area divided by the “total peptide”. Peak integrations and masses for each extract are listed in Supplementary Table 6. All analysis is done in python 3.5 using pandas, scipy, numpy, and matplotlib libraries.
Peptide secondary metabolites are common in nature and have diverse functions, from antibiotics to cross-kingdom signaling, that have been harnessed as pharmaceuticals. Their amino acid structure simplifies binding to protein targets and they have constraints and chemical modifications that enhance affinity, stability and solubility. A method to design large libraries of modified peptides in Escherichia coli and screen them in vivo to identify those that bind to a target-of-interest was developed in this Example. Constrained peptide scaffolds were produced using modified enzymes gleaned from microbial RiPP (ribosomally synthesized and post-translationally modified peptides) pathways and diversified to build large libraries. RiPP binding to a target protein leads to the intein-catalyzed release of a 6 factor. This circuit was used to drive a selection, which could evaluate 108 variants in a single experiment. This was applied to the discovery of a 1625 Da constrained peptide (AMK-1057) that binds with 990±5 nM affinity to the SARS-CoV-2 Spike receptor binding domain (RBD), a potential therapeutic target.
Bacteria and fungi secrete modified peptides that can act on eukaryotic cells by binding to cell-surface proteins, inhibiting enzymes or affecting protein-protein interactions [1-3]. They can be produced by large non-ribosomal peptide synthases or encoded by genes and post-translationally modified (RiPPs) [4-8]. As pharmaceuticals, cyclic peptides are approved for the treatment of cancer, inflammation, and infection and increasing numbers are entering all phases of clinical development for diverse indications [9-12]. They have shown promise for blocking viral entry into human cells [13,14]. For example, the FDA-approved HIV therapeutic Enfuvirtide is a 36 amino acid (aa) linear peptide that binds to a transmembrane glycoprotein; however, it suffers from rapid proteolysis, thus requiring twice daily injections [15]. Crosslinking HIV-1 mimetic peptides makes them proteolytically-stable, acid-resistant, and orally bioavailable [16].
Discovering peptides that bind to a therapeutic target requires methods to: (1) create massive pools of chemical diversity, and (2) identify hits in an efficient manner. Synthetic chemistry can be used to create libraries of modified peptides, including cycles and glycosylation, which are screened individually in assays that can be automated [17-24]. Encoding the peptide with its genetic material facilitates the panning for those that bind to a target, for example, using fluorescence activated cell sorting (FACS) [18-20,23, 25-29]. This can be done through yeast display, mRNA-peptide fusions and phage display, which have been used to find modified peptides that are antibiotics or bind human therapeutic targets [26, 29-36]. Cyclization can be performed enzymatically, chemically, or with split inteins, which are naturally occurring proteins that splice two separately-expressed peptides into an excised intein and a product [37,38].
If target binding can be linked to gene expression, this can be used to drive a reporter for screening or a marker that allows cells to survive a selection. The classic example is a two-hybrid system where a “bait” protein fused to DNA-binding domain recruits the “prey” protein fused to an activator that turns on a promoter when bound [39-44]. This can be used to find molecules that disrupt the bait-prey interaction, which has been applied to the discovery of linear peptides that are antivirals or block cancer signaling or progression [40, 45-47]. An E. coli version led to the discovery of a cyclized RiPP μM inhibitor of the p6-UEV protein-protein interaction necessary for HIV budding [41,44]. Protein-protein interactions have also been detected using split inteins where, upon binding, a reporter (epitope, fluorescent protein or a factor) is released, but this has not been applied to molecular discovery [48,49].
Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, is dependent upon cell recognition and entry mediated by the interaction of viral surface glycoprotein (Spike) receptor binding domain (RBD) and host receptor angiotensin-converting enzyme 2 (ACE2) (
A genetic circuit in E. coli that responds when a modified peptide binds to a single bait protein was developed and used to drive a selection to identify hits that bind to the SARS-CoV-2 Spike RBD. Libraries of modified peptides were produced by artificially combining enzymes from microbial RiPP pathway that introduces thioether-based macrocycles to constrain the peptide (Paenibacillus polymyxa PapB) [62-64] and vary the unmodified core residues. Each candidate RiPP was fused to a C-terminal intein and one half of a split a factor (RiPP-NpuC-σC) and modified in this context (
The genetic circuit described in this Example converts a binding event into a transcriptional response (e.g., the expression of a reporter protein;
It is important that the expression of the σN-NpuN and NpuC-σC fragments, in the absence of bait or peptide, does not induce the circuit. The experiments described above were repeated for these fragments lacking bait or peptide. At maximal expression of the σN-NpuN and NpuC-σC fragments (lacking bait or peptide), the output promoter was activated, albeit at 8-fold lower activity than when the bait and peptide were included (
The inducible range of the sensor was then determined when either the bait or peptide were swapped to disrupt the interaction. When a peptide based on the N-terminal residues 19-56 of ACE2 (ACE2*), which does not bind to the Mdm2* target, was used, the fluorescent output of the circuit dropped 15-fold. Similarly, when the target peptide was swapped to be residues 328-533 of the SARS-CoV-2 Spike protein (RBD) 51, to which PMI does not bind, the output dropped 93-fold (
The peptide needs to be able to be modified by RiPP enzymes in the context of its fusion to C-terminal NpuC-σC (
A preliminary experiment was performed to ensure that an enzyme of interest could modify a large fraction of core sequences in a library without being impacted by the C-terminal fusion (
The genetic system used for the selections, involving nine genes, is shown in
The libraries of modified peptides were constructed using oligo synthesis with NNK codons at the varied residues and cloned into a low copy pSC101 plasmid. The library was transformed using electrocompetence, which was found to limit the library size to 108 per transformation. Then, multiple rounds of positive selection were performed. The details for each library are described further below. When a RiPP binds the target, expression of Cat is increased, thereby conferring chloramphenicol resistance to the host cell (
The library was based on the simplified PapB-modified core structure shown in
The 20 hits from this library were codon optimized, re-synthesized and cloned into the RiPP-npuC-σC plasmid and re-assessed in freshly transformed cells. Testing of newly synthesized constructs was intended to eliminate any cheater behavior that may have arisen throughout the selection process. These constructs were transformed into selection strains containing cognate modifying enzymes and either Spike RBD or Mdm2* as bait, with the latter intended to measure off-target binding. The circuit output was measured using flow cytometry under the same growth conditions and inducer concentrations used for the selections. The core sequence VCKYGEWCEIVEI (SEQ ID NO: 24) demonstrated a strong transcriptional output and 14-fold specificity for the Spike RBD as bait over Mdm2* (
The core sequence VCKYGEWCEIVEI (SEQ ID NO: 24) underwent liter-scale production, cleavage and purification (
Co-expression of this peptide fusion with PapB in E. coli Marionette X (NEB Express derivative) cells followed by Ni-NTA affinity purification yielded tagged and modified pap2c_1. A peak corresponding to unmodified peptide was also detected. Dialysis of Ni-NTA purified peptide, TEV cleavage, solid phase extraction (SPE) and semiprep HPLC purification led to the isolation of three peptides: leader (yield: 200 μg/L), unmodified core (640 μg/L) and modified core (360 μg/L).
High resolution LCMS analysis of both modified (expected m/z: 1625.7338; observed m/z: 1625.7332) and unmodified (expected m/z: 1627.7494; observed m/z: 1627.7484) peptide showed a mass shift corresponding to formation of a single cycle, despite the library being based on a two-cycle scaffold (
In vitro binding experiments were then performed using Expi293F human cell-derived and purified RBD. Bio-layer interferometry (BLI) was used to measure the affinity of AMK-1057 to Spike RBD as 990±5 nM (
This Example demonstrates a technique to capture modified peptides that bind to a single target protein. There are several advantages over a two-hybrid screen, including that the binding target does not have to be known (or be a protein) or able to be expressed in a heterologous host, and hits will not be discovered against the “wrong” target (in this case, to human ACE2). As a relevant example of the importance of this capability, clinically relevant betacoronaviruses to date share a common Spike protein for host recognition, but the host receptor is not known a priori [50]. This allows for the search for binders to begin before their cellular targets have been fully elucidated. The libraries provided in this Example are based on natural products built with RiPP enzymes, a family that has been rapidly growing and for which there are many interesting chemical conversions, including halogenation, backbone N-methylation, and β-amino acid formation [80-82]. Larger biologics, such as antibodies, can have problems with stability and are limited in possible modes of delivery [59]. In contrast, cyclic peptides can exhibit improved stability, be cell-permeable thereby enabling access to intracellular antiviral targets, and be suitable for administration via inhalation [83-86].
Using this approach, a small peptide binder to SARS-Cov Spike RBD was identified. At ˜1600 Da, AMK-1057 is a size that is common for peptide secondary metabolites and approaches the threshold for the commonly used definition of a small molecule (˜900 Da) [9]. At <1 μM binding, AMK-1057 is in the higher range of natural RiPPs binding to their target (e.g., lassomycin at 0.41 μM, microcin J25 at 2 μM) and some peptidic drugs (e.g., vancomycin at ˜1 μM) [87-89]. As the first hit to emerge from a selection, it is ripe for further optimization through additional diversification and medicinal chemistry. This work represents a critical initial step of drug discovery. Putative therapeutics targeting viral fusion need to progressively tested in assays for the blockage of viral entry into cell lines [90-93], followed by animal models [92,93]. A human organ-chip has also been developed to screen repurposed drug compound collections that inhibit viral pseudoparticles expressing SARS-CoV-2 Spike from infecting human lung epithelial cells [94]. Combining molecular diversity creation using the method provided herein with a selection circuit in the same cell enables massive libraries to be evaluated to populate these pharmaceutical discovery pipelines with binders to a target-of-interest with minimal biochemical information.
E. coli NEB 10-beta (C3019I, New England BioLabs, Ipswich, Mass., USA) was used for all routine cloning. E. coli Marionette-Clo 70 was used for all selection experiments. E. coli Marionette-X, a Marionette-compatible derivative of NEB Express (C2523I, New England BioLabs, Ipswich, Mass., USA) was used for large-scale peptide expression experiments. TB (T0311, Teknova, Hollister, Calif., USA) supplemented with 0.4% glycerol (BDH1172-4LP, VWR, OH, USA) was used for peptide expression and modification. 2xYT liquid media (B244020, BD, Franklin Lakes, N.J., USA) and 2xYT+2% agar (B214010, BD, Franklin Lakes, N.J., USA) plates were used for routine cloning and strain maintenance. SOB liquid media (S0210, Teknova, Hollister, Calif., USA) was used for making competent cells. SOC liquid media (B9020S, New England BioLabs, Ipswich, Mass., USA) was used for outgrowth. Unless noted otherwise, cells were induced with the following chemicals: cuminic acid (268402, Millipore Sigma, Saint Louis, Mo., USA) added as 1000× stock (200 mM) in EtOH or DMSO; 3-oxohexanoyl-homoserine lactone (3OC6-AHL) (K3007, Millipore Sigma, Saint Louis, Mo., USA) added as a 1000× stock (1 mM) in DMSO; anhydrotetracycline (aTc) (37919, Millipore Sigma, Saint Louis, Mo., USA) added as a 1000× stock (100 PM) in DMSO; isopropyl β-D-1-thiogalactopyranoside (IPTG) (I2481C, Gold Biotechnology, Saint Louis, Mo., USA) added as 1000× stock (1 M) in water. Cells were selected with the following antibiotics: carbenicillin (carb, C-103-5, Gold Biotechnology, Saint Louis, Mo., USA) added as 1000× stock (100 mg/mL in H2O); kanamycin (kan, K-120-10, Gold Biotechnology, Saint Louis, Mo., USA) as 1000× stock (50 mg/mL in H2O); spectinomycin (spec, S-140-5, Gold Biotechnology, Saint Louis, Mo., USA); and chloramphenicol (Cm, C-105-5, Gold Biotechnology, Saint Louis, Mo., USA). Liquid chromatography was performed with Optima Acetonitrile (A996-4, Thermo Fisher Scientific, MA, USA) and water (Milli-Q Advantage A10, Millipore Sigma, Saint Louis, Mo., USA) supplemented with LCMS Grade Formic Acid (85178, Thermo Fisher Scientific). The following solvents/chemicals were also used: Ethanol (V1001, Decon Labs, King of Prussia, Pa., USA), Methanol (3016-16, Avantor, Center Valley, Pa., USA), Ammonium bicarbonate (A6141 Millipore Sigma, Saint Louis, Mo., USA), dimethyl sulfoxide (DMSO) (32434, Alfa Aesar, Ward Hill, Mass., USA), Imidazole (IX0005, Millipore Sigma, Saint Louis, Mo., USA), sodium chloride (X190, VWR, OH, USA), sodium phosphate monobasic monohydrate (20233, USB Corporation, Cleveland, Ohio, USA), sodium phosphate dibasic anhydrous (204855000, Acros, N.J., USA), guanidine hydrochloride (50950, Millipore Sigma, Saint Louis, Mo., USA), tris (75825, Affymetrix, Cleveland, Ohio, USA), TCEP (51805-45-9, Gold Biotechnology, Saint Louis, Mo., USA), and EDTA (0.5M stock, 15694, USB Corporation, Cleveland, Ohio, USA). DNA oligos and gBlocks were ordered from Integrated DNA Technologies (IDT) (San Francisco, Calif., USA).
Plasmids pTHSS-1282 and pAMK-267 were constructed from the parental pTHSS-1193 backbone, which has a pSC101 origin variant (var 2) and ampicillin resistance [95]. Plasmids pTHSS-1282 and pAMK-267 contain a flexible linker sequence (GSSRGGKGGPGGRGGVGGGGGIGG (SEQ ID NO: 113)) between the peptide/sfGFP and NpuC regions. Plasmids pAMK-925, pTHSS-2132, pAMK-866, and pAMK-870, were constructed from the parental pTHSS-1458 backbone, which has a colE1* origin variant and a kanamycin resistance marker [95]. All plasmids carrying modifying enzymes were constructed from the parental pEG01_189 backbone and contain a p15A origin of replication and spectinomycin resistance [78]. The parental backbone pTHSS-2012, which has a p15a origin and spectinomycin resistance was used for additional cloning experiments [95]. The plasmid pTHSS-1282 that contains the P20_992 promoter expressing sfGFP was constructed from pTHSS-1193. The plasmids pAMK-926 and pTHSS-2137 that contain the PLux promoter expressing NpuC-σC and PMI-NpuC-σC, respectively, were constructed from pTHSS-2012. The plasmids pAMK-925 and pTHSS-2132 that contain the PTac promoter expressing σN-NpuN and residues 17-124 of Mdm2 (Mdm2*)-σN-NpuN, respectively, were constructed from pTHSS-1458. The plasmid pAMK-870 that contains the constitutive PJ23105 promoter expressing Mdm2*-σN-NpuN and the P20_992 promoter expressing CAT-sfGFP was constructed from pTHSS-1458. The plasmid pAMK-866 that contains the constitutive PJ23105 promoter expressing 328-533 of the SARS-CoV-2 Spike protein (RBD)-σN-NpuN and the P20_992 promoter expressing CAT-sfGFP was constructed from pTHSS-1458. The peptide cloning plasmid pAMK-267, constructed from pTHSS-1193, contains the PLux promoter upstream of an RBS-His tag-SapI-sfGFP-SapI-NpuC-σC where the sfGFP gene can be replaced by a peptide gene through Type IIs assembly methods using the enzyme SapI (NEB). The RBS from pAMK-267 was chosen from a library of RBS variants upstream of a His tag-PMI-NpuC-σC that was tuned for co-expression with constructs containing the PJ23105 promoter expressing Mdm2*-σN-NpuN. The N-terminal His tag in pAMK-267 was left in place to provide a constant 11 aa for consistent levels of expression between different peptide sequences. The plasmid pAMK-670 that contains the PLux promoter expressing PMI-NpuC-σC was constructed from pAMK-267. The plasmid pAMK-857 that contains the PLux promoter expressing N-terminal residues 19-56 of ACE2 (ACE2*)-NpuC-σC was constructed from pAMK-267. The pTHSS-1193 and pTHSS-1458 backbones have origin variants that alter their copy numbers, making them approximately equivalent to a p15a backbone. Genes encoding Npu intein, PMI, Mdm2*, ACE2*, and RBD were synthesized as gBlocks. The ECF20_992 gene was sourced from a previous publication [65].
Fluorescence characterization was performed on a BD LSR Fortessa flow cytometer with HTS attachment (BD, Franklin Lakes, N.J., USA). Samples were prepared by diluting overnight cultures 1:400 by adding 0.5 μl of cell culture into 200 μl of PBS containing 1 mg/mL Kan. All samples were run in standard mode at a flow rate of 0.5 μl/s. Fluorescence measurements were made using the blue (488 nm) laser and all data was derived from the FITC-A channel (PMT voltage of 400 V). The FSC and SSC voltages were 650 V and 270 V, respectively. At least 30,000 events were collected for each sample and the Cytoflow Python package was used for downstream analysis. Gating was completed by fitting a 2D Gaussian function to the FSC and SSC distributions and excluding all events greater than three standard deviations from the mean. When presented, the median value is used.
Strains of E. coli Marionette Clo harboring a combination of plasmids pTHSS-1282, pTHSS-2132, and pTHSS-2137 or pTHSS-1282, pAMK-925, and pAMK-926 were used for assessing intein splicing with or without PMI-Mdm2* induced association, respectively. Strains were grown in 1 mL of LB+ antibiotics for 20 hr in a deep well 96-well plate (1896-2000, USA Scientific, FL, USA) at 30° C., 900 rpm in an Infors HT Multitron Pro (Infors USA, MD, USA). Cultures were then diluted 1:100 into fresh 1 mL of LB+ antibiotics and serial 1:10 dilutions of inducers (IPTG, 10−3-103 μM; 3O6-AHL, 10−3-103 nM) for 20 hr in a deep well 96-well plate at 30° C., 900 rpm in the Multitron Pro. 0.5 μl of saturated cell culture were then diluted into 200 μl of PBS containing 1 mg/mL kan for cytometry analysis.
To assay for protein-protein mediated splicing the following plasmid combinations were transformed into E. coli Marionette Clo and fluorescence was measured via cytometry: pAMK-866/pAMK-670 (RBD/PMI); pAMK-866/pAMK-857 (RBD/ACE2*); pAMK-870/pAMK-670 (Mdm2*/PMI); pAMK-870/pAMK-857 (Mdm2*/ACE2*). Strains were grown in 1 mL of LB+ antibiotics for 20 hr in a deep well 96-well plate at 30° C., 900 rpm in a Multitron Pro. Cultures were then diluted 1:100 into fresh 1 mL of LB+ antibiotics+1 μM 3O6-AHL (full induction of peptide plasmid) for 20 hr in a deep well 96-well plate at 30° C., 900 rpm in the Multitron Pro. 0.5 μl of saturated cell culture were then diluted into 200 μl of PBS containing 1 mg/mL Kan for cytometry analysis.
The Pap library was designed with diversity at the ends and middle of the peptide and included either glutamate or aspartate as a cyclization partner, for a final sequence design of “XCXXX[D/E]XCXXX[D/E]X (SEQ ID NO: 114)”. Using the degenerate nucleotide sequences “NNK” to encode any amino acid and “GAW” for aspartate or glutamate, a library of 1012 peptides encoded by 1014 unique codon sequences was generated. The library of plasmids lbAMK-103, which contains the PLux promoter expressing the Pap library-NpuC-σC was constructed from pAMK-267. The pap library was amplified from pEG03_283 using degenerate oligonucleotides oAMK-915/916 (IDT). Gel purification was used to isolate the 124 bp amplicon, which was then cloned into pAMK-267 using the type IIS restriction enzyme SapI (NEB).
Linear insert and plasmid were mixed at a 1:1 molar ratio (200 fmol each) along with 10 μl 1×DNA ligase buffer, 2 μl T4 DNA ligase (HC) (20 U/μl) (M1794, Promega, Madison, Wis., USA) and 4 μl SapI in 100 μl total volume. Reactions were cycled 25 times for 2 min at 37° C. and 5 min at 16° C. then incubated for 30 min at 50° C., 30 min at 37° C., and 10 min at 80° C. in a DNA Engine cycler (Bio-Rad, Hercules, Calif., USA). An additional 2 μl SapI was then added, and the assembly was incubated for 1 h at 37° C. Assemblies were then purified using Zymo Spin I columns (Zymo Research, Irvine, Calif., USA). Library assemblies were initially transformed into electrocompetent NEB 10βE. coli (C3020K, NEB, Ipswich, Mass., USA). 1.5×107 colony forming units (CFU)/mL were observed for lbAMK-103. Total transformants were estimated by colony counting after 107-fold dilution and plating of liquid outgrowths on selective media.
The initial, unselected papA library was transformed and plated to resolve individual colonies. A set of 19 random colonies were picked and sequenced via colony PCR. Of the 19 sequenced colonies, 18 were properly assembled. These 18 library members were then assessed for post-translational modification via LCMS. The 9 unmodified and 5 modified library sequences were then aligned and WebLogos generated (weblogo.berkeley.edu/logo.cgi) with default parameters, except without small sample correction.
Selection of Pap Library lbAMK-103.
Assembled library of plasmids lbAMK-103 was transformed into an electrocompetent Marionette Clo strain harboring the PapB modifying enzyme plasmid, pEG06_044, and the selection plasmid, pAMK-866 (all non-assembly transformation steps were >1×108 efficiency). A 1 mL of liquid outgrowth of library transformants was diluted 1:50 in TB+Carb/Kan/Spec+1 μM 3OC6-AHL and 100 μM cumate to induce peptide+modifying enzyme, and grown at 30° C., 250 r.p.m. for 20 h. For the first round of selection, cultures were then diluted 1:100 in TB Carb/Kan/Spec+1 μM 3OC6-AHL and 100 μM cumate+300 μM Cm and grown at 30° C., 250 r.p.m. for at least 20 h (until cultures were saturated). A 0.5 μL aliquot of was taken for cytometry analysis and 2 mL of culture was also taken to harvest plasmid. A 5 μL sample of purified plasmid was stored for NGS analysis and the rest was digested with 1 μL SapI (NEB) for 1 hour at 37° C. to remove the background pEG06_044/pAMK-866 plasmid. The selected lbAMK-103 plasmid was then re-transformed into the strain of electrocompetent E. coli Marionette Clo strain harboring the PapB modifying enzyme, pEG06_046, and the selection plasmid, pAMK-866. The selection process was repeated once more with a Cm concentration of 800 μM and then once more with a Cm concentration of 1200 μM.
Library construction for NGS was performed using the protocol for “KAPA Hyper Prep Kits with PCR Library Amplification/Illumina series” (KK8504, Roche). First, miniprepped library plasmids were amplified with Q5 polymerase (#M0492L, New England BioLabs, Ipswich, Mass., USA) with the primers oAMK-946/947 (Pap library) and oAMK-997/998 (Tgn/Lyn library). A 150 bp band was isolated via gel extraction. Indexed adapters were ligated and reamplified with 10 cycles of PCR. Gel extraction was then used to isolate the resultant 260 bp PCR product. Sample concentrations were calculated using a BioAnalyzer on a High Sense DNA chip (5067-4626, Agilent). Samples were diluted to 2 nM, denatured, and further diluted to 10 μM, with a 10% phiX spike in. Samples were run on a HiSeq 2500 using HiSeq v2 reagents for Paired End Clustering and a 200 cycle SBS kit (PE-402-4002 and FC-402-4021, Illumina). Forward and reverse reads were both 110 cycles, with an 8-cycle single index read. Base-calling and demultiplexing were performed using the bcl2fastq software (Illumina) with default settings. After basecalling and indexing, sequences were identified and aligned using the leader sequence and then binned by sequence.
Validation of sequences from NGS. Hit peptides from NGS were resynthesized as gBlocks (IDT). These gBlocks were used as template for PCR to introduce SapI restriction sites compatible for re-cloning into the pAMK-267 library backbone. Newly reconstructed library members were transformed into Marionette-Clo cells containing modifying enzyme and selection plasmids and were then plated on media containing Carb/Kan/Spec. Individual transformants were then cultured in TB+Carb/Kan/Spec in a deep well 96-well plate (1896-2000, USA Scientific, FL, USA) and incubated overnight (Multitron Pro, 30° C., 900 rpm). These cultures were then subcultured 1:100 in TB+Carb/Kan/Spec either fully induced (1 μM 3OC6-AHL, and 100 μM cumate) or uninduced and incubated for 20 hr (Multitron Pro, 30° C., 900 rpm) before taking 0.5 μL for standard flow cytometry analysis.
Potential peptide hit gBlocks were cloned into the peptide expression plasmid, pEG03-119 78 using their flanking SapI restriction sites. The peptide and modifying enzyme plasmids were co-transformed into E. coli Marionette-X, streaked onto 2xYT agar with carb/spec and incubated at 30° C. overnight. Individual colonies were used to inoculate 20 mL of LB in a 125 mL shake flask and incubated overnight at 30° C. and 250 rpm in an Innova44 (Eppendorf, N.Y., USA). A 5 mL aliquot of overnight starter culture was diluted in 500 mL total volume TB with carb/spec in Fernbach flasks and grown at 30° C. and 250 rpm until reaching OD600 of 0.8-1.0, at which point 1 mM IPTG and 200 μM cumate were added. Induced cultures were grown for a further 20 h at 30° C. and 250 rpm and then centrifuged (4,000 g, 4° C., 10 min) in a Sorvall RC 6+ centrifuge (Thermo Fisher Scientific, MA, USA). Pellets were resuspended in 30 mL lysis buffer (5 M guanidinium hydrochloride, 300 mM NaCl, 10 mM imidazole, 50 mM sodium phosphate, pH 7.5), and freeze-thawed twice (frozen in −80° C. freezer; thawed in innova44 incubator at 30° C., 250 rpm). Cell lysates were centrifuged (Eppendorf 5424, 20,000 g, 18° C., 45 min) in a Sorvall RC 6+ centrifuge (Thermo Fisher Scientific, MA, USA) and the peptides affinity purified via gravity-flow using 3 mL resin-bed volume of Ni-NTA agarose resin (88223, Thermo Fisher Scientific, MA, USA), following manufacturer instructions, using 2 resin-bed volumes water and lysis buffer for column equilibration, 4 resin-bed volumes of wash buffer (5 M guanidinium hydrochloride, 300 mM NaCl, 25 mM imidazole, 50 mM sodium phosphate, pH 7.5), 4 resin-bed volumes of elution buffer (5 M guanidinium hydrochloride, 300 mM NaCl, 250 mM imidazole, 50 mM sodium phosphate, pH 7.5). Eluate from Ni-NTA purification was then subjected to solid-phase extraction (SPE) using Strata-XL 500 mg tubes (8B-S043-HCH, Phenomenex, CA, USA). The solid phase was first conditioned with 4 bed volumes of methanol and then water. Eluate was then loaded, washed with 8 bed volumes of 10 mM NH4CO3, and eluted with 8 bed volumes of 1:1 acetonitrile:aqueous 10 mM NH4CO3. Solvent was removed via lyophilization at −80 C for 24-48 hours. To cleave the SUMO and leader peptide from the core, the extracted peptide was resuspended in 20 mL TE buffer and 100 μl of 20 mg/mL TEV protease and incubated overnight at room temperature with slow orbital shaking. The cleaved peptides were then desalted using a Strata-X PRO 500 mg SPE tubes (8B-S536-HCH, Phenomenex, CA, USA). The solid phase was first conditioned with 4 bed volumes of methanol and then water. Eluate was then loaded, washed with 8 bed volumes of 10 mM NH4CO3, and eluted with 8 bed volumes of 1:1 acetonitrile:aqueous 10 mM NH4CO3. Solvent was removed via lyophilization at −80 C for 24-48 hours. After solvent removal, a 5 mL aliquot of the mixture resuspended in 10:90 acetonitrile:water was injected into a Agilent Technologies 1260 Infinity system HPLC (Agilent Technologies, Santa Clara, Calif.) and separated using a 150 mm×10 cm Aeris PEPTIDE XB-C18 column (100 Å, 5 μm) at a flow rate of 2 mL/min. Separation was carried out with a gradient program, with 0.1% formic acid as solvent A and acetonitrile with 0.1% formic acid as solvent B. The % B was held at 25% for 3 minutes, then increased to 50% over the next 17 minutes. The eluent was passed through a diode array detector (DAD) and absorbance at 270 nm was recorded. Detected peaks were collected using an Agilent G1364B Fraction Collector and again solvent was removed via lyophilization at −80 C for 24-48 hours. Samples were resuspended in 1 mL of 1:1 acetonitrile:aqueous 10 mM NH4CO3 in pre-weighed 2 mL microcentrifuge tubes (Eppendorf) and solvent was removed via lyophilization at −80 C for 24-48 hours. Yields were measured by comparing mass of empty tubes to tubes containing lyophilized powder.
All chromatography was performed using the mobile phases ACN (acetonitrile supplemented with 0.1% formic acid and 0.1% water) and water (supplemented with 0.1% formic acid). The “QTOF” was an Agilent 1260 Infinity II liquid chromatograph with binary pump configured in low-dwell volume mode and column oven set to 40° C., coupled to an Agilent 6545 QTOF mass spectrometer equipped with an Agilent electrospray ionization (ESI) source. ESI source parameters are 350° C. gas temperature, 12 L/min gas flow, 30 psig nebulizer pressure, 350° C. sheath gas temperature, 8 L/min sheath gas flow, 3000 V capillary voltage, 1000 V nozzle voltage, 135 V fragmentor voltage, 15 V skimmer voltage, 600 V Oct 1 RF Vpp; the mass spectrometer was run in MS mode with reference mass enabled and tuned in positive mode with standard mass range (3200 m/z) and 2 GHz extended dynamic range. QTOF analysis was performed with a Phenomenex Aeris PEPTIDE XB-C18 2.6 μm 50 mm×2.1 mm column. The flow rate was set at 0.5 mL/min and 5 μl sample was injected. The gradient used was 20% ACN for 0.5 min, 20% to 55% ACN over 5.5 min, 55% to 90% ACN over 0.5 minutes, 90% ACN for 1.5 min, with 0.8 min re-equilibration. Accurate mass predictions of peptides were generated using the online resource, ChemCalc [96].
Assays were performed on an Octet Red (ForteBio) instrument at 30° C. with shaking at 1,000 rpm. Ni-NTA biosensors (18-5101, ForteBio, Bohemia, N.Y., USA) were hydrated in 1× kinetics buffer (diluted from 10× buffer; 18-5032, ForteBio, Bohemia, N.Y., USA) for 30 min before the measurement. Expi293F human cell-derived and purified SARS-CoV-2 RBD (RBD296-531) was loaded at 10-20 μg/mL in 1× Kinetics Buffer for 300 s prior to baseline equilibration for 180 s in 1× kinetics buffer. Association reactions of the peptide to RBD296-531 were carried out in 1× kinetics buffer at various concentrations in a two-fold dilution series from 80 mM to 1.25 mM was carried out for 900 s. Then dissociation reactions were observed for 900 s. Response data were generated using ForteBio data analysis software.
AMK-1057, a small peptide binder, was evaluated for cell competition between the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and the human ACE2 receptor. RBD incubated with and without AMK-1057 was mixed with ACE2 cells, washed, and quantified via flow cytometry (
Bio-layer interferometry was used to assay AMK-1057 competition for binding to RBD in the presence of B38 and CR3022 antibodies as well as purified ACE2 for the purpose of mapping what region of the RBD AMK-1057 may bind. RBD binding to AMK-1057 was not affected by the presence of B38 (
The human microbiome harbors substantial biosynthetic potential for specialized metabolites with roles in host-microbe and microbe-microbe interactions. Analysis of genomic sequence data from the Human Microbiome Project shows an untapped source of post-translationally modified peptides, a class of molecule demonstrated to have important effects on human health and disease. Genome mining approaches, wherein DNA sequences are synthesized de novo and heterologously expressed in chassis organisms, can be leveraged to access the molecules encoded in human microbiome sequence data. However, robust methods for large-scale interrogation of sequence space through DNA synthesis and heterologous expression have yet to be developed. Here, 78 biosynthetic gene clusters were selected for post-translationally modified peptides from a diverse set of human microbiome strains from all niches of the human body. Production of peptides was shown in a format suitable for screening their biological activity and novel molecules with unique spectra of antimicrobial activity against members of the human microbiome and pathogenic bacteria of clinical significance were identified. This work demonstrates that large-scale genome mining of peptidic natural products and functional assaying for their biological activity is possible through a DNA sequence-to-molecule pipeline.
Revealing how the human microbiome affects health at a mechanistic level will continue to be critical in understanding disease and developing new therapies1. Discovery and characterization of specialized metabolites (small molecules, peptides) is of particular interest due to their important role in biological systems and pharmaceutical potential as standalone agents or effectors in cell-based therapeutics2. Traditional approaches to the isolation of specialized metabolites from the human microbiome have been hampered by access to putative producing organisms and difficulties in eliciting production. A number of bioinformatics tools are now available to parse ever-increasing DNA sequence data, annotate biosynthetic gene clusters, and assign basic molecular predictions3. These tools make possible a “sequence-to-molecule” approach, wherein mining DNA sequence databases, selecting gene clusters for DNA synthesis, and heterologous expression can yield specialized metabolites of value. However, the rate of molecular production is orders of magnitude behind in silico identification of the encoding DNA. Production of molecules is handicapped by difficulties with the large size of many gene clusters, appropriate heterologous production hosts, and standardized approaches for their purification as well as structural elucidation4.
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of specialized metabolite particularly abundant in human microbiome DNA sequence data5-7. RiPPs are defined by a conserved biosynthetic logic wherein a precursor peptide (comprised of a “leader” and “core” region) is ribosomally produced, the core subsequently altered by modifying enzymes that often recognize sequence motifs in the leader, then ultimately processed and exported (
As of 2015, 100 lanthipeptides had been discovered from microbes14 and half that number of lasso peptides15. New computational approaches to RiPP genome mining have yielded impressive advances in the discovery of RiPP subclasses and scaffolds but actual molecular discovery is relatively low (˜1-5 molecules per report) and functional assaying is either absent or narrow in scope16-21. The flexible biosynthesis afforded by RiPPs has also led to a number of innovative strategies for generating large libraries around a given peptide scaffold linked to a functional output. These include libraries based on the lasso peptide microcin J2522, the thiopeptide thiocillin23, and the lanthipeptides nisin, prochlorosin, haloduracin, and lacticin 48124-28. While of outstanding value, these approaches all require specialized assays and selections and do not exploit specific biological activities afforded by natural evolution. There is a need for higher throughput approaches to purify, express, and structurally annotate RiPPs that can then be tested in diverse functional assays. Here, an E. coli-based expression system was used to mine 78 RiPP gene clusters to generate 23 new lanthipeptides and lasso peptides from the human microbiome. The established pipeline was able to go from DNA sequence information to a structurally and functionally annotated molecule in relatively high-throughput. These 23 structurally annotated RiPPs, combined with 7 RiPPs with unknown modification, were demonstrated to have unique scaffolds and spectra of antimicrobial activity when tested against a large panel of human microbiome-associated strains. A subset of these RiPPs were shown to possess activity against multidrug resistant (MDR) clinical isolates of human pathogens, including vancomycin resistant Enterococcus and methicillin-resistant Staphylococcus aureus. This provides a robust method for accessing a vast and underexplored chemical space of the human microbiome.
AntiSMASH29 was used to identify 2,233 RiPP gene clusters from 2,231 genomes of the Human Microbiome Project (HMP)30. BiG-SCAPE31 was then used to generate a sequence similarity map of these gene clusters to visualize the abundance of different subclasses of RiPP (
In addition to the defining biosynthetic enzymes described above (LanBC, LanM, LanK for lanthipeptides; LasBC for lasso peptides), “tailoring enzymes” that further chemically diversify peptides can be encoded in gene clusters. Tailoring enzymes can modify bioactivity of peptides and have promise in functioning as catalysts for engineering RiPPs35 so open reading frames encoding putative tailoring enzymes were included in the mining workflow. Novel tailoring enzymes were not identifiable by existing in silico methods so a script was developed to identify and count the presence of all protein family (pfam) domains found in gene clusters annotated by AntiSMASH. These pfam counts were converted to relative abundance by dividing raw counts by the presence of core biosynthetic enzymes (lanBC/M/K; lasBC) and rank-ordered to profile prevalence of certain pfam domains in each subclass of RiPP investigated here (
78 gene clusters were selected from 68 diverse organisms spanning 6 classes and occupying airway, gastrointestinal (GI) tract, oral, skin, and urogenital (UG) tract microbiomes (
Streptococcus pneumoniae
Streptococcus pneumoniae
Dolosigranulum pigrum
Dolosigranulum
pigrum Aguirre et al.
Staphylococcus caprae
Staphylococcus
caprae (ATCC ® 55133 ™)
Staphylococcus capitis
Staphylococcus capitis
Staphylococcus epidermis
Staphylococcus epidermidis
Streptococcus infantarius
Streptococcus infantarius
Bacteroides
—
dorei
Bacteroides
—
faecis
Bacteroides
—
thetaiotaomicron
Bifidobacterium
—
adolescentis
Bifidobacterium
—
longum
Citrobacter
—
amalonaticus
Enterococcus
—
avium
Enterococcus
—
durans
Enterococcus
—
mundtii
Leuconostoc
—
lactis
Paeniclostridium
—
sordellii
Parabacteroides
—
distasonis
Parabacteroides
—
goldsteinii
Pediococcus
—
acidilactici
Ruthenibacterium
—
lacta-
tiformans
Sellimonas
—
intestinalis
Veillonella
—
dispar
Streptococcus sobrinius
Streptococcus sobrinius 6715
Streptococcus mitis
Streptococcus
mitis Andrewes and Horder
Streptococcus gordonii
Streptococcus gordonii Kilian
Streptococcus mutans
Streptococcus mutans UA159
Rothia dentocariosa
Rothia dentocariosa (Onishi)
Corynebacterium striatum
Corynebacterium
striatum (Chester) Eberson
Micrococcus luteus
Micrococcus
luteus (Schroeter) Cohn
Staphylococcus aureus
Staphylococcus aureus subsp.
aureus ATCC-19685
Staphylococcus hominis
Staphylococcus
hominis subsp. hominis Kloos
Streptococcus dysgalactiae
Streptococcus dysgalactiae
Streptococcus sanguinis
Streptococcus
sanguinis White and Niven
Lactobacillus crispatus JV-V01
L. crispatus JV-V01
Lactobacillus jensenii ATCC
L. jensenii ATCC 25258
Lactobacillus gasseri ATCC
L. gasseri ATCC 33323
Acinetobacter baumannii
Aspergillus fumigatus
Campylobacter jejuni
Candida albicans
Enterococcus faecalis
Enterococcus faecium
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
Salmonella Typhimurium
Staphylococcus aureus
E. coli is an Effective Chassis Organism for Genome Mining of RiPPs
Application of this workflow to the selected gene clusters resulted in the detection and subsequent structural annotation of 18 lanthipeptides and 5 lasso peptides (
7 lanthipeptide clusters generated retention/mass shifts in the presence of modifying enzymes but mass shifts weren't consistent with known modification patterns. Of particular interest were several producing strains that showed modifications via retention time/mass shift when putative tailoring enzymes were expressed (
A diverse selection of producing organisms were selected from which to mine lanthipeptide sequences for heterologous expression and whether gene clusters from particular genera were more or less suitable for expression in E. coli was investigated. To this end, a taxonomic tree of all lanthipeptide-producing organisms (with E. coli BL21 for reference) selected for this study was generated. Strains from which that successfully produced a RiPP were highlighted to detect trends (
96-well microtiter growths (2×1 mL TB media) were purified and processed and optimal conditions for assaying biological activity were considered. Agar plate-based assays that demonstrate antimicrobial activity via zones of inhibition are an ideal method since compounds do not suffer dilution as in liquid-based readouts of optical density. Microtiter-purified RiPPs were initially tested against a subset of human microbiome-associated strains (Staphylococcus aureus, Streptococcus infantarius, Streptococcus dysgalactiae, Pediococcus acidilactici, Pseudofalnovifractor spp., and Bacteroides faecis) to assess this plate-based method and several producing strains (sAMK-287, sAMK-687, sAMK-691) showed varying zones of inhibition against this initial test set of indicator strains (
To streamline functional assaying, 96-well microtiter growths were optimized for a large collection of indicator strains sourced from a variety of niches found in the human microbiome (Table 11). The large-scale antimicrobial profiling of 30 SPE purified RiPPs (including both peptides that were confirmed via the structural annotation pipeline as well as putative modified peptides) showed that 8/30 demonstrated unique antimicrobial “fingerprints”. Of these active peptides, 7/8 could be grouped either through a common source cluster (AMK-286, 287, 916; AMK-917, 1009, 1010) or a common structural scaffold (AMK-417, 687, 691). The eighth, AMK-720, is an uncharacterized modified peptide that showed exceptionally broad antimicrobial activity. The structure and biosynthesis of AMK-720 are still under investigation, but structure-function relationships for the other three groups of peptides are described below.
The type II lanthipeptides AMK-286, AMK-287, and AMK-916 were based on genes from an oral strain of Streptococcus and share identical modification profiles (
The lasso peptides AMK-917, 1009, and 1010 are from an oral strain of Rothia dentocariosa and exhibit conserved primary amino acid sequence about the lariat structure, with some degeneracy (
Amino acid sequence alignments showed that AMK-417, 687, and 691 belong to the same family of RiPPs as lacticin 481 and the structural annotation was consistent with a similar cyclization pattern (
Streptococcus_pneumoniae_
Streptococcus_sp._
Streptococcus_sp._
Rothia dentocariosa
Ruminococcus
flavefaciens
Ruminococcus
flavefaciens
Clostridium spp.
Corynebacterium_
matruchotii_ATCC_14266
Gardnerella_vaginalis_
Rothia_dentocariosa_
Clostridium_botulinum_
Myroides odoratimimus
Lactobacillus iners
Streptococcus pyogenes
Streptococcus pyogenes
Mobiluncus mulieris
Streptococcus
pneumoniae
Lactobacillus delbrueckii
Streptococcus agalactiae
Staphylococcus caprae
Myroides odoratimimus
Streptococcus sanguinis
Streptococcus agalactiae
Streptococcus agalactiae
Eubacterium sp.
Dolosigranulum pigrum
Streptococcus_sp._
Rothia aeria F0474
Enterococcus faecalis
Sphingobium
yanoikuyae
Rothia aeria FO474
Rothia aeria F0474
Based on the large antimicrobial activity dataset, four peptides (AMK-287, 417, 687, and 691) were selected to characterize their activity against clinical isolates of MDR pathogens. SPE-purified peptides from liter scale fermentations were used to profile dose-dependent killing of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and Streptococcus pneumoniae (
Attempts to address, at a mechanistic level, the dynamics of the microbiome commonly rely on a kind of “forward genetics” approach (start with a phenotype and move toward microbial genetic determinants)1. Here instead, a group of molecules were systematically assessed to functionally profile them for their potential to shape the microbiome. RiPPs sourced from the human microbiome may hold specific advantages as narrow spectrum antimicrobials for combating MDR pathogens. Traditional antibiotics can exacerbate the evolution of resistance or are causative of disease outright through their broad-spectrum activity disrupting the human microbiome54. Lanthipeptides with antimicrobial activity act primarily through targeting the cell envelope55, which is an attractive strategy to sidestep resistance mechanisms linked to enzymatic modification and efflux. Cyclic peptide natural products (or mimetics) targeting the bacterial outer envelope are being investigated and studied in clinical trials, including those active against Gram-negative pathogens56-58.
Several of the molecules discovered here serve as excellent scaffolds for further examining structure-activity relationships of the variable cyclic regions. The 96-well microtiter expression pipeline enables both rapid assessment of biosynthetic constraints for modifying enzyme/peptide pairs and functional assaying against indicator organisms of interest. Modifying enzymes that are associated with multiple substrate peptides can also serve as effective biocatalysts for selections of modified peptides with de novo activity25,39. Cell-free expression approaches, as demonstrated for unmodified bacteriocins59, offer a useful method for initial activity testing, but scalable production routes must be considered. Systematic heterologous expression and engineering of RiPP gene clusters (e.g., as provided herein) addresses the production issue and also advances peptides' potential as cell-based effectors in living therapeutics60. Emerging technology for the delivery of genetic programs to diverse bacteria61,62 coupled with responsive, in situ peptide production to sidestep unfavorable pharmacokinetic properties63 further highlights the therapeutic potential of peptides.
Semi-purified RiPPs were produced directly from sequence information without downstream assay constraints from as little as 2 mL microwell fermentations. Expression of RiPPs scaled well to liter volumes and methods were established for rapidly purifying and generating screening plates of peptides dissolved in an organic solvent/water mixture. These plates can be frozen, stored, and treated in similar fashion to small molecule libraries, enabling their broad assaying. The enumeration of medium-sized natural products in this format is of particular value since, compared to small molecules, they are under sampled in most natural products screening collections64. Medium-sized modalities exhibit greater efficacy in binding 15 to and disrupting non-enzymatic function of macromolecular targets65.
The scale at which RiPP gene clusters were constructed, expressed, and characterized in this study is unprecedented but precludes widespread, in-depth structural characterization. The application of high-resolution tandem mass spectrometry to characterize post-translationally modified peptides, however, is an acceptable level of structural annotation, as evidenced by comparable studies9-11, 39-45. The workflows described here enable discovery, prioritization, and optimization of a limited number of molecules, which can be scaled in production volume for more rigorous structural and functional characterization as appropriate.
In summary, a platform was developed for streamlined genome mining of RiPP gene clusters. Rapid assessment of modification through 96-well expression, purification, and LC-MS analysis enabled small molecule and novel enzyme discovery. Application of this pipeline toward genome mining of the human microbiome yielded constrained peptides with unique antimicrobial fingerprints when tested against a large subset of strains from the human microbiome. These molecules were shown to be active against MDR bacterial pathogens. Systematic discovery and functional profiling of human microbiome-derived antimicrobials able to selectively target endogenous microflora and pathogens has significant potential for both engineering the microbiome and developing therapeutics to address antimicrobial resistance.
Strains, media, and chemicals. E. coli NEB 10-beta (C3019I, New England BioLabs, Ipswich, Mass., USA) was used for all routine cloning. E. coli NEB Express (C2523I, New England BioLabs, Ipswich, Mass., USA) and E. coli Marionette-X, a Marionette-compatible derivative of NEB Express were used for liter-scale peptide expression experiments. TB (T0311, Teknova, Hollister, Calif., USA) supplemented with 0.4% glycerol (BDH1172-4LP, VWR, OH, USA) was used for peptide expression and modification. 2xYT liquid media (B244020, BD, Franklin Lakes, N.J., USA) and 2xYT+2% agar (B214010, BD, Franklin Lakes, N.J., USA) plates were used for routine cloning and strain maintenance. Other media include Tryptic Soy Broth (TSB; BD211825, BD, Franklin Lakes, N.J., USA), Brain Heart Infusion (BHI; BD237500, BD, Franklin Lakes, N.J., USA),
Lactobacilli MRS broth (MRS; BD288130, BD, Franklin Lakes, N.J., USA), and Sabouraud Dextrose Broth (SDB; BD288130, BD, Franklin Lakes, N.J., USA). SOB liquid media (S0210, Teknova, Hollister, Calif., USA) was used for making competent cells. SOC liquid media (B9020S, New England BioLabs, Iwsich, Mass., USA) was used for outgrowth. Unless noted otherwise, cells were induced with the following chemicals: cuminic acid (268402, Millipore Sigma, Saint Louis, Mo., USA) added as 1000× stock (200 mM) in EtOH or DMSO; 3-oxohexanoyl-homoserine lactone (3OC6-AHL) (K3007, Millipore Sigma, Saint Louis, Mo., USA) added as a 1000× stock (1 mM) in DMSO; anhydrotetracycline (aTc) (37919, Millipore Sigma, Saint Louis, Mo., USA) added as a 1000× stock (100 μM) in DMSO; isopropyl β-D-1-thiogalactopyranoside (IPTG) (I2481C, Gold Biotechnology, Saint Louis, Mo., USA) added as 1000× stock (1 M) in water; Sodium salicylate (S3007, Millipore Sigma, Saint Louis, Mo., USA), N-(3-Hydroxytetradecanoyl)-DL-homoserine lactone (3OC14-AHL; 51481, Millipore Sigma, Saint Louis, Mo., USA. Cells were selected with the following antibiotics: carbenicillin (carb, C-103-5, Gold Biotechnology, Saint Louis, Mo., USA) added as 1000× stock (100 mg/mL in H2O); kanamycin (kan, K-120-10, Gold Biotechnology, Saint Louis, Mo., USA) as 1000× stock (50 mg/mL in H2O); and spectinomycin (spec, S-140-5, Gold Biotechnology, Saint Louis, Mo., USA). Liquid chromatography was performed with Optima Acetonitrile (A996-4, Thermo Fisher Scientific, MA, USA) and water (Milli-Q Advantage A10, Millipore Sigma, Saint Louis, Mo., USA) supplemented with LCMS Grade Formic Acid (85178, Thermo Fisher Scientific). The following solvents/chemicals were also used: Ethanol (V1001, Decon Labs, King of Prussia, Pa., USA), Methanol (3016-16, Avantor, Center Valley, Pa., USA), Ammonium bicarbonate (A6141 Millipore Sigma, Saint Louis, Mo., USA), dimethyl sulfoxide (DMSO) (32434, Alfa Aesar, Ward Hill, Mass., USA), Imidazole (IX0005, Millipore Sigma, Saint Louis, Mo., USA), sodium chloride (X190, VWR, OH, USA), sodium phosphate monobasic monohydrate (20233, USB Corporation, Cleveland, Ohio, USA), sodium phosphate dibasic anhydrous (204855000, Acros, N.J., USA), guanidine hydrochloride (50950, Millipore Sigma, Saint Louis, Mo., USA), tris (75825, Affymetrix, Cleveland, Ohio, USA), TCEP (51805-45-9, Gold Biotechnology, Saint Louis, Mo., USA), and EDTA (0.5M stock, 15694, USB Corporation, Cleveland, Ohio, USA), dimethyl formamide (A13547, Alfa Aesar, MA, USA), defibrinated sheep blood (R54012, Thermo Fisher Scientific, MA, USA), hemin (51280, Sigma Aldrich), vitamin K1 (V3501, Sigma Aldrich), and L-cysteine (C7532, Sigma Aldrich). DNA oligos and gBlocks were ordered from Integrated DNA Technologies (IDT) (San Francisco, Calif., USA).
Computational detection and clustering of RiPP gene clusters. Genome datasets for projects “HMP1” and “HMP2” were obtained from the Human Microbiome Project online portal. These 2,229 genomes were used as the database for running AntiSMASH 4.0 using default parameters with ClusterFinder-based border predictions 29. Output from this analysis was analyzed using BiG-SCAPE with distance cut-off filters of 0.2, 0.4, 0.6, 0.8, and 1.0. The resulting similarity network matrices were visualized with Cytoscape and distance cutoff of 0.8 chosen for
Peptide expression in 96-well plates and purification. Plasmids were transformed into either E. coli NEB Express or E. coli Marionette-X using 30 μL of competent cells and 1 μL of each plasmid being transformed in a PCR strip tubes (1402-4700, USA Scientific, FL, USA or 951020401, Eppendorf, N.Y., USA). Transformations were incubated on ice (20-30 min), heat shocked (42° C., 30 sec), and incubated on ice again (5 min). Cells were then transferred to a deep well 96-well plate (1896-2000, USA Scientific, FL, USA) with 120 μL of SOC. After outgrowth (Multitron Pro, 1 hr, 30° C.) in an Infors HT Multitron Pro (Infors USA, MD, USA), 900 μL LB was added with appropriate antibiotics (at 1.1× for 1× final concentration) and incubated (Multitron Pro, 30° C., 900 r.p.m.) until all wells reached saturation (12-30 hours). Overnight cultures were diluted 1:100 into 1 ml TB in deep well plates. After 3 hours incubation (Multitron Pro, 30° C., 900 r.p.m.), appropriate inducer was added (1 μl IPTG or 1l1 IPTG and 1 μl cumate), and cultures were incubated for 20 hours (Multitron Pro, 30° C., 900 r.p.m.). To purify the peptides, the 96-well plates were centrifuged (Legend XFR, 4,500 g, 4° C., 20 min), pellets were resuspended in 600 μL lysis buffer, and freeze-thawed twice (frozen at −80° C.; thawed in Multitron Pro at 37° C., 900 r.p.m). Cell lysates were centrifuged (Legend XFR, 4,500 g, 4° C., 60 min) and peptides affinity purified using His MultiTrap TALON plates (29-0005-96, GE Life Sciences, Marlborough, Mass., USA), following manufacturer instructions, using 1×600 μL water and 2×600 μL lysis buffer for column equilibration, 2×600 μL wash buffer, and 1×200 μL elution buffer.
Liter-scale RiPP expression and purification. Glycerol stocks of strains generated from 96-well transformations were used to inoculate 20 mL of LB in a 125 mL shake flask and incubated overnight at 30° C. and 250 rpm in an Innova44 (Eppendorf, N.Y., USA). A 5 mL aliquot of overnight starter culture was diluted in 500 mL total volume TB with carb/spec in Fernbach flasks and grown at 30° C. and 250 rpm until reaching OD600 0.8-1.0, at which point 1 mM IPTG and 200 μM cumate are added. Induced cultures were grown for a further 20 h at 18° C. and 250 rpm and then centrifuged (4,000 g, 4° C., 10 min) in a Sorvall RC 6+ centrifuge (Thermo Fisher Scientific, MA, USA). Pellets were resuspended in 30 mL lysis buffer (5 M guanidinium hydrochloride, 300 mM NaCl, 10 mM imidazole, 50 mM sodium phosphate, pH 7.5), and freeze-thawed twice (frozen in −80° C. freezer; thawed in innova44 incubator at 30° C., 250 rpm). Cell lysates were centrifuged (20,000 g, 12° C., 45 min) and the peptides affinity purified via gravity-flow using 3 mL resin-bed volume of Ni-NTA agarose resin (88223, Thermo Fisher Scientific, MA, USA), following manufacturer instructions, using 2 resin-bed volumes water and lysis buffer for column equilibration, 4 resin-bed volumes of wash buffer (5 M guanidinium hydrochloride, 300 mM NaCl, 25 mM imidazole, 50 mM sodium phosphate, pH 7.5), 4 resin-bed volumes of elution buffer buffer (5 M guanidinium hydrochloride, 300 mM NaCl, 250 mM imidazole, 50 mM sodium phosphate, pH 7.5). Eluates were diluted to 30 mL with lysis buffer, transferred to Spectra/Por 3 RC Dialysis Membrane Tubing 3500 Dalton MWCO (132725, Spectrum, CA, USA) and dialyzed overnight at room temperature in 1× phosphate buffered saline (PBS; 6505-4L, CalBiochem, CA, USA). Dialyzed solutions were centrifuged (4,000 g, 4° C., 10 min) to remove any precipitate. To cleave the SUMO and leader peptide from the core, TCEP (1 mM final concentration) and 3 mg of TEV protease (30 mg lyophilizate, Gene and Cell Technologies, CA, USA) were added and tubes incubated overnight at room temperature with slow orbital shaking. Cleaved peptide solutions were centrifuged (4,000 g, 4° C., 10 min) to remove any precipitate and then desalted using a Strata-X PRO 500 mg SPE tube (8B-S536-HCH, Phenomenex, CA, USA). The solid phase was first conditioned with 4 bed volumes of methanol and then water. Eluate was then loaded, washed with 8 bed volumes of water, and eluted with 8 bed volumes of 1:1 acetonitrile:water+0.1% formic acid. Solvent was removed via lyophilization at −80 C for 24-48 hours.
Liquid chromatography/mass spectrometry. All chromatography was performed using mobile phases ACN (acetonitrile supplemented with 0.1% formic acid and 0.1% water) and water (supplemented with 0.1% formic acid). LC-MS was performed on one of two mass spectrometers: “QQQ” is an Agilent 1260 Infinity liquid chromatograph with binary pump configured in low-dwell volume mode, high-performance autosampler chilled to 18° C., and column oven, coupled to an Agilent 6420 QQQ mass spectrometer equipped with an Agilent electrospray ionization (ESI) source; nitrogen gas is supplied by a Parker Nitroflowlab and ESI source parameters are 350° C. gas temp at 12 L/min flow rate, 15 psi nebulizer voltage, 4000 V capillary voltage, 135 V fragmentor voltage, and 7 V cell accelerator voltage. “qTOF” is an Agilent 1260 Infinity II liquid chromatograph with binary pump configured in low-dwell volume mode and column oven set to 40° C., coupled to an Agilent 6545 qTOF mass spectrometer equipped with an Agilent electrospray ionization (ESI) source; nitrogen gas is building supplied and ESI source parameters are 350° C. gas temperature, 12 L/min gas flow, 30 psig nebulizer pressure, 350° C. sheath gas temperature, 8 L/min sheath gas flow, 3000 V capillary voltage, 1000 V nozzle voltage, 135 V fragmentor voltage, 15 V skimmer voltage, 600 V Oct 1 RF Vpp; the mass spectrometer was run in MS mode with reference mass enabled and tuned in positive mode with standard mass range (3200 m/z) and 2 GHz extended dynamic range. When using the QQQ, analysis was done with a Phenomenex Aeris PEPTIDE XB-C18 2.6 □m 50 mm×2.1 mm column with column oven set to 40° C. Flow rate was 0.6 ml/min. Gradient was 10% ACN for 0.5 min, 10% to 60% ACN over 6 min, 60% to 90% ACN over 1 min, 90% ACN for 1 min, with 1 min re-equilibration. The mass spectrometer was run in positive mode, 500-2000 m/z range with a 300 ms scan time. Injections were 5 □L (as a starting point, injection volumes were occasionally adjusted depending on the yield of the 96-well prep). When using the qTOF, analysis was done with a Phenomenex Aeris PEPTIDE XB-C18 2.6 □m 50 mm×2.1 mm column. Flow rate was set at 0.5 ml/min. The flow rate was set at 0.5 mL/min and 5 μL sample was injected. The gradient used was 10% ACN for 1.0 min, 10% to 70% ACN over 5.0 min, 70% to 90% ACN over 0.5 minutes, 90% ACN for 1.0 min, with 1.0 min re-equilibration. Injections were 5 μL (as a starting point, injection volumes were occasionally adjusted depending on the yield of the 96-well prep).
Peptide screening plate prep. Lyophilized liter-scale preps were resuspended in 540 μL DMF and vortexed for 5 seconds. To this was added 3060 μL of H2O and the mixture was vortexed for 5 seconds to make a solution of peptide in 15% DMF. All mixtures were centrifuged (Legend XFR, 4,000 g, 4° C., 10 min) to remove any insoluble material and then split into 2 96-well 2 mL plates. From this, 12 μL of each peptide was aliquoted into 290 96-well screening plates (3788, Corning), which were then used for downstream LC-MS/MS analysis and functional assay screening. Plates were covered and kept at −20° C. for up to one year.
LC-MS/MS data acquisition. All chromatography was performed using the mobile phases ACN (acetonitrile supplemented with 0.1% formic acid and 0.1% water) and water (supplemented with 0.1% formic acid). MS/MS data were acquired on an Agilent 1260 Infinity II liquid chromatograph with binary pump configured in low-dwell volume mode and column oven set to 40° C., coupled to an Agilent 6545 qTOF mass spectrometer equipped with an Agilent electrospray ionization (ESI) source. Nitrogen gas is building-supplied and ESI source parameters are 350° C. gas temperature, 12 L/min gas flow, 30 psig nebulizer pressure, 350° C. sheath gas temperature, 8 L/min sheath gas flow, 3000 V capillary voltage, 1000 V nozzle voltage, 135 V fragmentor voltage, 15 V skimmer voltage, 600 V Oct 1 RF Vpp; the mass spectrometer was run in MS mode with reference mass enabled and tuned in positive mode with standard mass range (3200 m/z) and 2 GHz extended dynamic range. For this analysis, 4 peptide screening plates were thawed and resuspended in a total of 100 □L PBS/DMF mixture. To this mixture, TCEP was added to a final concentration of 1 mM. Samples were split in two and NEM (12.5 mM final concentration) was added to one group of samples to label free cysteine residues. For the targeted MS/MS, 4 spectra/s were sampled with fixed collision energies of 30, 45, 60, and 75 V. A narrow isolation width (1.3 m/z) and observed monoisotopic mass (exact masses found in Supplementary Fig. xx) was used for fragmentation of each peptide. Sample analysis was performed with a Phenomenex Aeris PEPTIDE XB-C18 2.6 □m 50 mm×2.1 mm column. The flow rate was set at 0.5 mL/min and 5 □L sample was injected. The gradient used was 10% ACN for 1.0 min, 10% to 70% ACN over 5.0 min, 70% to 90% ACN over 0.5 minutes, 90% ACN for 1.0 min, with 1.0 min re-equilibration. Accurate mass predictions of peptides were generated using the online resource, ChemCalc 68. Indicator strain growth. Indicator strains were grown using the annotated media. The following specialized media mixtures were used: TSB supplemented with 5% defibrinated sheep blood (TSBb) and BHI supplemented with hemin, vitamin K1, and L-cysteine (BHIs). To make BHIs, 10 mL of hemin solution (50 mg hemin, 1 mL 1 N NaOH, 100 mL H2O, filter sterilized) and 200 μL of diluted vitamin K1 solution (150 μL vitamin K1 solution, 30 mL 95% ethanol, filter sterilized) were added to sterile 1 L sterile BHI supplemented with 0.5 g L-cysteine. Agar plates of all media types were generated by addition of 2% agar. For strains sourced from OpenBiome and individual labs, strains were first purified by streaking on agar media plates. For strains sourced from ATCC and CDC, product protocols were followed to activate lyophilizates and strains were grown on agar plates of the annotated media type. All strains were grown on solid media until uniform colonies were observed. Individual colonies were used to inoculate sterile 96-well microtiter plates of the corresponding media type. Once wells reached sufficient density (24-72 hours of growth, see additional culturing conditions below), liquid glycerol stocks were generated by the addition of 500 μL culture and 500 μL 50% glycerol. Multiple glycerol stock plates were generated and frozen at −80° C. for subsequent assaying described below.
Antimicrobial assays. All materials were additionally sterilized by exposure to UV light for 10 minutes in laminar flow cabinet. Glycerol stocks of microbiome strains were subcultured in liquid media. Strains were grown for 24-48 hours, diluted 1:200 into fresh media, and 100 μL added to thawed peptide screening plates previously generated. Compounds were aliquoted in wells C1-E12 with wells B1-B12 and F1-F12 containing 15% DMF controls. Additional media was added to wells surrounding the assay wells to mitigate evaporation. All growth plates contained wells B1-B12 with a no growth control (15% DMF plus 100 μL media) and wells F1-F12 with a growth control (15% DMF plus 100 μL diluted culture). Plates were manually inspected for sufficient control growth after 24 or 48 hours and optical density measured using a The OD600 was measured using a Synergy H1 Hybrid Reader (8041000, BioTek). Automated plate shaking was found to be insufficient to break up pellets formed by some strains and therefore all pellets were manually broken up by mild pipetting with care taken to not introduce bubbles. Residual growth was calculated by measuring the OD600 of all plate wells. All measurements were done in triplicate on three separate days. Dilution series experiments were performed as above with new compound preps. Compounds were mixed with media at 4× the final concentration. Serial two-fold dilutions into the same media composition generated a compound dilution series at 2× the final assay concentration. Diluted indicator cultures were added 1:1 to this mixture to generate a 1× compound concentration in all wells.
Combining peptide sequence constraints for peptide-modifying enzymes, such as those identified through methods described in the previous examples and shown in
As a proof of principle, the modification patterns of three enzymes were combined and analyzed to develop core and leader sequence motifs. As shown in
After analyzing the core and leader sequence variant possibilities, a chimeric leader and hybrid core motif were identified combining the options for LynD, PlpXY, and ThcoK modifications (
Similar methods were applied to additional combinations of modification enzymes: (a) ThcoK and LynD; (b) PadeK and LynD; (c) LynD and LasF; (d) PalS, PlpXY and PadeK; (e) LasF and PalS; (f) PlpXY, ThcoK and LynD; (g) PadeK and PalS; and (h) ThcoK and PalS. A selection of peptides were identified for these combinations (
RiPPs (ribosomally-synthesized and post-translationally modified peptides) are a class of pharmaceutically-relevant natural products expressed as precursor peptides before being enzymatically processed into their final functional forms. Bioinformatic methods have illuminated hundreds of thousands of RiPP enzymes in sequence databases and the number of characterized chemical modifications is growing rapidly; however, it has proven difficult to functionally express them in a heterologous host. A major challenge is peptide stability, which is addressed in this Example by design of a RiPP stabilization tag (RST) based on a small ubiquitin-like modifier (SUMO) domain that can be fused to the N- or C-terminus of the precursor peptide and proteolytically removed after modification. This is demonstrated to stabilize a set of eight RiPPs representative of diverse phyla without interfering with the activity of associated modifying enzymes. Further, using Escherichia coli for heterologous expression, a common set of media and growth conditions were identified in which 24 modifying enzymes, representative of diverse chemistries, were shown to be functional. The high success rate and broad applicability of this system enables RiPP discovery through high-throughput “mining” as well as retrosynthesis through the artificial combination of enzymes from different pathways to create a desired non-natural peptide.
Metagenomics has led to a deluge of microbial genomes, leading to high-throughput efforts to “mine” the molecules made by organisms by rebuilding pathways and screening for functions-of-interest[1-3]. Because these genes are gleaned from sequence databases, the organism or genomic DNA may not be available, thus necessitating the use of DNA synthesis and a heterologous host to obtain the chemical product[4-6]. RiPPs (ribosomally-synthesized and post-translationally modified peptides) are a potentially rich source of functional diversity that are encoded in gene clusters as a precursor peptide that is enzymatically modified before being proteolytically released[7-14]. Because the peptidic product is made by the ribosome, rather than by a large megasynthase, the probability of successful heterologous expression was determined to be high. However, expressed peptides are often unstable in vivo, and post-translational modifying enzymes may not function in new contexts[15-17]. As a result, only a small fraction of the thousands of known RiPP pathways have been explored[13].
RiPPs are classified by the chemical modifications made to the peptide. Some are defined by cyclization chemistry, including lanthipeptides (lanthionine macrocyclizations), thiopeptides ((4+2) cycloaddition of dehydrated serine/threonine), lasso peptides (N-terminal macrocyclization with asp/glu), graspetides (lactone/lactam macrocyclizations), bottromycin (macrolactamidine macrocyclization), ranthipeptides (Non-Cα thioether macrocyclizations), pantocins (glutamate crosslink), and sactipeptides (sactionine macrocyclizations)[7, 14]. Others are defined by individual modifications, like glycocins (side chain glycosylation), microcin C (aminoacyl adenylation or cytidylation), comX (indole cyclization and prenylation), sulfatyrotide (tyrosine sulfation), spliceotide (β-amino acids from backbone splicing), and cyanobactins (N-terminal proteolysis). Precursor peptide organization varies between RiPP classes. Modifying enzymes can either bind to a leader/follower sequence in the precursor peptide or directly modify the core. The core consists of 2 to over 50 amino acids and there can be multiple cores in one precursor peptide[17-20]. Leader peptides range from 7 to over 80 amino acids and can recruit multiple modifying enzymes that can have overlapping binding sequences[21-23]. The diversity in chemistry and genetic encoding complicates the creation of general engineering tools that can be systematically used for mining efforts across RiPP classes.
Tools have been developed to aid heterologous production, including multi-plasmid inducible systems and exploration of E. coli, various Streptomyces strains, and Microvirgula aerodentrificans as expression hosts[17, 30-33]. In vitro methods have also been used to engineer production of new molecules or study biosynthesis[34-37]. Gene cluster regulation may not function properly in a new host. To overcome this, the precursor peptide and modifying enzymes can be cloned and expressed separately[17, 33]. However, precursor peptides have been observed to often be unstable due to host proteases, thus necessitating the use of stabilization tags[15, 16, 24]. Large tags must be removed before peptide modifications can be observed by mass spectrometry, such as in the case of maltose binding protein (MBP, 45 kD), green fluorescent protein (GFP, 27 kD) and glutathione-S-transferase (GST, 26 kD)[38]. In contrast, the small ubiquitin-like modifier tag (SUMO, 12 kD) is smaller, thus allowing modifications to be observed prior to its removal. Further, it can be removed using SUMO protease immediately after purification without desalting[39], which simplifies its use in high-throughput formats. SUMO has been used for expression of both eukaryotic and prokaryotic antimicrobial peptides in E. coli [40-42] as well as a post-translationally modified lanthipeptide from Lactococcus[43] and a xenorceptide from Xenorhabdus[44].
Here, a RiPP Stabilization Tag (RST) was developed. The RST is a SUMO-based tag for high-throughput RiPP production and was demonstrated to work with diverse classes and modifying enzymes. Versions were made for fusion to the N- or C-terminus of the precursor peptide. Each version contains a HIS6 tag to enable purification in 96-well format. TEV and thrombin protease cleavage sites were included for the N- and C-terminal versions, respectively. Optimized E. coli inducible systems[45] were used to express tagged precursor peptides and modifying enzymes from separate plasmids. The ability for the RST to stabilize the peptide was validated by testing precursor peptides from 9 RiPP classes. As an example, it was demonstrated that the B. halodurans antibiotic peptide haloduracin A1/A2 can be expressed in E. coli and completely modified while attached to the RST, and further that the peptide is functional upon proteolytic cleavage of the RST. Fifty (50) precursor peptides were tested with 47 modifying enzymes and 39 peptides were identified that were expressed as RST fusions, and 24 were identified that were able to be modified with the RST attached. This Example demonstrates the broad applicability of the RST tag for high-throughput mining efforts that span RiPP classes and modifying enzyme chemistries. In addition, these enzymes were all expressed in the same heterologous host (E. coli) under uniform culture conditions and induction times. This provides a roadmap for selecting those enzymes that can be artificially combined to build retrosynthetic pathways for producing non-natural RiPP molecules with desired properties.
Two versions of the RiPP stabilization tag (RST) were designed to allow fusion to either the N- or C-terminus (termed RSTN and RSTC, respectively) of a precursor peptide (
A two-plasmid system was used to separately express the precursor peptide and modifying enzyme, thus enabling combinations to be tested rapidly through co-transformations (
Expression and purification protocols were first developed for low-throughput growth in 250 ml flasks in LB media. The tagged precursor peptide and modifying enzyme were induced simultaneously. After induction with 1 mM IPTG and 200 μM cumate (for PCymR*) or 10 μM 3OC6-AHL (for PLuxB), cultures were grown at 18° C. for 20 hours with shaking. Then, the peptide was purified using immobilized metal affinity chromatography (IMAC) and analyzed using LC-MS.
An example of the production of a modified peptide in flasks is shown in
Next, RST stabilization of diverse precursor peptides across RiPP classes was tested (
The ability for RSTN* to stabilize the unmodified peptides was tested. Expression was measured in the absence of modifying enzymes to account for any stabilization affect that arises from peptide modification. Expression and purification were performed at the 250 mL flask scale, as described above. First, precursor peptide expression when fused only to a N-terminal HIS6 tag was evaluated. This tag led to only three of nine peptides being detected by LC-MS (
Next, the production of a biologically-active product was evaluated using the expression system provided herein. Modifications were directed at an RST-fused peptide, after which the tag was cleaved and the activity of the product tested. Haloduracin was selected, a two-component lanthipeptide that had previously been expressed and purified from E. coli and shown to have antibiotic activity[34]. Genes encoding haloduracin A1 and haloduracin A2 peptides fused to RSTN were synthesized, as were genes encoding corresponding HalM1 and HalM2 modifying enzymes from Bacillus subtilis (
A high-throughput 96-well system for expression and purification was developed, which was tested using haloduracin. Cultures were grown in 2 mL of TB media in deep well plates (two 1 mL wells for each peptide), where they are each induced with 1 mM IPTG/200 μM cumate for 20 hours at 30° C. with shaking. The cells were lysed, affinity-purified and desalted using solid phase extraction, all in 96-well format. Then, the samples were treated with TEV protease to remove RSTN and the leader peptide, and desalted again to concentrate the core peptide (
To assay for antimicrobial activity, the cleaved and desalted core peptides were resuspended in 50 μL 1:1 methanol:water. Bacillus subtilis PY79 was used as indicator strain and was spread on a LB-agar surface, on which 5 μL of either or both haloduracins or a solvent control was added. Individually, the haloduracin peptides showed limited activity (
A set of 47 modifying enzymes and their cognate 50 precursor peptides was collated from the literature. The complete list of pathways and enzymes is provided in Table 13 and Table 14, and the subset ultimately found to be active in this Example is provided in Table 15. The selected modifying enzymes are representative of 13 bacterial RiPP classes from diverse genera and catalyze 22 different chemical transformations, including glycosylation, radical carbon-carbon bond focpation and cysteine heterocyclization. The precursor peptide and modifying enzyme genes were codon optimized for E. coli and synthesized, or amplified when the source DNA was available, and cloned into the two-plasmid system. The precursor peptides were tagged with RSTN, except for macrocyclization of lasso peptides, which were fused to RSTC. The plasmids containing the modifying enzymes and precursor peptides were co-transformed into E. coli NEB Express.
Lentzea kentuckyensis
Burkholderia thailandensis E264
Streptomyces albus
Asticcacaulis excentricus
Caulobacter sp. K31
Caulobacter segnis
Paenibacillus dendritiformis C454
Thermobacillus composti KWC4
Paenibacillus polymyxa CR1
Streptomyces sp. Amel2xC10
Listeria monocytogenes SLCC2540
Aeribacillus pallidus 8
Bacillus amyloliquefaciens DSM7
Bacillus subtilus
Pantoea agglomerans
Xanthomonas oryzae
Pleurocapsa sp. PCC7319
Pleurocapsa sp. PCC7327
Carnobacterium maltaromaticum C2
S. globisporus subsp. globisporus
Bacillus cereus SJ1
Lactococcus lactis
Prochlorococcus MIT9313
Escherichia coli
Microbispora corallina
Streptomyces cinnamoneus
cinnamoneus DSM 40005
Bacillus halodurans C-125
Staphylococcus epidermidis
Anabaena sp. PCC7120
Plesiocystis pacifica
Microcystis aeruginosa NIES843
Bacillus thuringiensis serovar
Prochloron spp.
Prochloron spp.
Microcystis aeruginosa NIES-88
Planobispora rosea
Bacillus subtilis subsp. spizizenii
Paenibacillus polymyxa ATCC 842
aMass shift listed is for a single modification. Enzymes can multiply-modify their peptide substrate, resulting in a total mass shift that is multiplied by the integer number of modifications performed.
The cultures were grown following the high-throughput protocol in 96-well plates. Both TB and LB media have been used previously to functionally express certain RiPPs in E. coli. The choice of media can impact the function of an enzyme; for example, radical S-adenosyl-L-methionine (rSAM) enzymes are more active in TB than LB, the latter requiring a reduction in shake speed and/or increased iron-sulfur cluster biosynthesis [22, 60, 61]. For applications requiring the high-throughput mining or the artificial combination of RiPP enzymes (retrosynthesis), it is desirable to have a single set of culture conditions. To this end, the ability for the enzymes to modify their precursor peptides was evaluated following the same culture conditions either in LB or TB (Table 15 and
The 25 modified peptides shown in Table 14 showed the exact mass change that was expected to result from the modification shown. However, some modifications could occur at different positions than the wild-type modification, leading to a different peptide with the same mass. In instances in which multiple modification products are possible, the addition of an RST could change where the modification occurs. To test for this outcome, several modifications were selected from different classes for evaluation by LC-MS/MS. The following were selected for structural annotation: PsnA2 macrolactonization by PsnB, and PapA sactionine macrocyclization by PapB. The precursor peptides were modified to contain a TEV cleavage site between the leader and core peptides. The modifying enzymes and precursor peptides were expressed following the high-throughput protocol, the RST and leader peptide removed using TEV protease, and the modified core analyzed with LC-MS/MS. Fragmentation of PsnA2 was observed between the core repeats, with each core repeat fragment mass corresponding to two lactone macrocyclizations per repeat, in agreement with previously published results[19]. Within each core repeat, MS/MS was not able to validate the cyclization topology within each core, which was previously determined by analyzing partially hydrolyzed modified peptide. Without using high collision energies, fragmentation products of PapA were only observed outside of predicted C-D ring structures, in agreement with published MS/MS spectra[61].
Of the enzymes tested, 23 of the 47 did not correctly modify a peptide when co-expressed in E. coli. Patterns based on the phylogeny from which the pathway was sourced were sought, noting that the sources spanned cyanobacteria, actinobacteria, proteobacteria, and firmicutes (
While the number of characterized RiPP enzymes is growing rapidly in the literature, the conditions under which each enzyme is characterized vary across studies. This poses a challenge for high-throughput screening efforts if the conditions have to be re-optimized for each pathway. This Example presents a side-by-side survey of recombinant RiPP enzymes in E. coli, using the same growth and induction methods. Further, this Example provides protocols for every step to be performed in 96-well plate format under conditions that are consistent with high-throughput screening platforms [2, 70-72]. The RSTs address the problem of precursor peptide stability, for which degradation and solubility are the dominant causes of unobservable product. Their use increases the probability that a pathway will be successfully expressed in a new host; in other words, they increase the “hit rate” of screening efforts. The RSTs do not interfere with the action of modifying enzymes, facilitate high-throughput purification and do not need to be removed prior to LC-MS analysis of modifications. Software was developed to rapidly analyze LC-MS data. Collectively, this presents a suite of tools that enable the high-throughput screening of RiPP pathways mined from sequence databases [13, 73, 74]. In this manuscript, the action of only a single enzyme at a time was investigated. To mine complete RiPP-encoding gene clusters, additional enzyme genes can either be assembled as operons or placed under the control of different inducible promoters (e.g., E. coli Marionette as described in the preceding Examples).
The fraction of enzymes found to be functional in E. coli under common conditions was surprisingly high, especially considering the diversity in the source genera and chemistries. The success rate was much higher than the successful transfer of other natural products genes, such as non-ribosomal peptide synthases, which also produce peptidic products. These results imply that RiPP enzymes can be combined from different sources to create synthetic pathways from which all the enzymes can be functionally expressed. Indeed, several examples have been published demonstrating the artificial combination of RiPP enzymes from different source species and pathways to make products not observed in nature [30, 75, 76]. Knowing that roughly half of RiPP enzymes are functionally compatible with E. coli dramatically expands the potential peptide chemical space that can be explored through the artificial mixing-and-matching of these enzymes. Fully enabling this requires a better understanding of the rules for designing precursor peptides that can be acted on by multiple modifying enzymes, such as the rules provided herein and in the preceding Examples. Collectively, these tools for the mining and de novo design of RiPPs enable the exploration of the vast universe of modified peptides for novel antibiotics, intercellular communication channels, and signaling molecules that influence animal and plant physiology.
Strains, plasmids, media, and chemicals. E. coli NEB 10-beta (C3019I, New England BioLabs, Ipswich, Mass., USA) was used for all routine cloning. E. coli BL21 (C2530H, New England BioLabs, Ipswich, Mass., USA) was used to characterize RSTs and linker variants in low-throughput (flask) cultures. E. coli NEB Express (C2523I, New England BioLabs, Ipswich, Mass., USA) was used to express all other experiments. All plasmids containing RST-fused purcursor peptide genes use a pSC101 origin variant (var 2) with ampicillin resistance[77]. All plasmids carrying modifying enzyme genes contain p15A origins of replication and kanamycin resistance. LB-Miller media (B244620, BD, Franklin Lakes, N.J., USA) or TB media (T0311, Teknova, Hollister, Calif., USA) supplemented with 0.4% glycerol (BDH1172-4LP, VWR, OH, USA) were used for peptide expression and modification. 2xYT liquid media (B244020, BD, Franklin Lakes, N.J., USA) and 2xYT+2% agar (B214010, BD, Franklin Lakes, N.J., USA) plates were used for routine cloning and strain maintenance. SOB liquid media (S0210, Teknova, Hollister, Calif., USA) was used for making competent cells. SOC liquid media (B9020S, New England BioLabs, Iwsich, Mass., USA) was used for outgrowth. Cells were induced with the following chemicals: cumate (cuminic acid) ≥98% purity from Millipore Sigma (268402, Millipore Sigma, Saint Louis, Mo., USA) added as 1000× stock (200 mM) in EtOH or DMSO; isopropyl β-D-1-thiogalactopyranoside (IPTG) ≥99% purity (I2481C, Gold Biotechnology, Saint Louis, Mo., USA) added as 1000× stock (1 M) in water or DMSO; 3OC6-AHL from Millipore Sigma (K3007, Millipore Sigma, Saint Louis, Mo., USA) added as a 1000× stock (10 mM) in DMF. Cells were selected with the following antibiotics: 50 μg/ml kanamycin (K-120-10, Gold Biotechnology, Saint Louis, Mo., USA); 100 μg/ml carbenicillin (C-103-5, Gold Biotechnology, Saint Louis, Mo., USA); 30 μg/ml chloramphenicol. Liquid chromatography was performed with Optima Acetonitrile (A996-4, Thermo Fisher Scientific, MA, USA) and water (Milli-Q Advantage A10, Millipore Sigma, Saint Louis, Mo., USA) supplemented with LC-MS Grade Formic Acid (85178, Thermo Fisher Scientific). DNA oligos and gblocks were ordered from Integrated DNA Technologies (San Francisco, Calif., USA).
Gene design. A list of plasmids and corresponding plasmid maps are provided in Table 16. Amino acid sequences of all modifying enzymes and peptides are provided in Table 17. Sequences of genetic parts and full plasmids are provided in Table 18 and Table 19.
Peptide expression/modification from flasks and purification. Plasmids were transformed into E. coli BL21, struck out on 2xYT agar with carbenicillin (or chloramphenicol for pEG3017) and kanamycin (if co-transforming modifying enzyme) and incubated (30° C., overnight). Individual colonies were used to inoculate 3 mL of LB media in a culture tube (352059, Corning, N.Y., USA) and incubated overnight (30° C., 250 r.p.m.) in an Innova44 (Eppendorf, N.Y., USA). Aliquots (500 l) were taken from the overnight cultures and subcultured into 50 mL of LB media in a 250 mL Erlenmeyer flask. After 3 hours incubation (Innova44, 30° C., 250 r.p.m.), IPTG and 3OC6-AHL (if inducing modifying enzyme) was added to final concentrations of 1 mM and 10 μM and cultures were incubated for 20 hours (Innova44, 18° C., 250 r.p.m.) (note: IPTG was not added for pEG3017, where the MBP-tagged peptide is constitutively expressed). The 50 mL cultures were transferred to a falcon tube (352070, Corning, N.Y., USA), centrifuged (4,500 g, 4° C., 20 min) in a Sorvall Legend XFR Centrifuge (Thermo Fisher Scientific, MA, USA), pellets were resuspended in 600 μl lysis buffer (5 M guanidinium hydrochloride, 300 mM NaCl, 50 mM sodium phosphate, pH 7.5), and freeze-thawed twice (frozen in −80° C. freezer; thawed in innova44 incubator at 30° C., 250 r.p.m). Cell lysates were centrifuged (Eppendorf 5424, 21,130 g, room temperature, 15 min) in an Eppendorf 5424 Centrifuge (Eppendorf, N.Y., USA) and the peptides affinity purified using His SpinTrap TALON columns (29-0005-93, GE Life Sciences (now Cytiva), Marlborough, Mass., USA), following manufacturer instructions, using 600 μL lysis buffer twice for column equilibration, loading 600 □L clarified lysate, two washes with 600 μL wash buffer (300 mM NaCl, 50 mM sodium phosphate, 5 mM imidazole, pH 7.5), and 200 μL elution buffer (300 mM NaCl, 50 mM sodium phosphate, 200 mM imidazole, pH 7.5) for elution. Purifications used an Eppendorf 5424 centrifuge.
Calculation of peptide molar masses. For large peptides/proteins, mass was calculated as described for ESIprot79: five consecutively charged m/z's (m1, m2, m3, m4, m5) were taken from the spectra and used to calculate the charge states (z1, z2, z3, z4, z5) for each of the peaks. For peaks m1 and m2, which have charge states, z1 and z2, where z2=z1−1 (peak 1 has one proton more than peak 2): z1=(m2−1)/(m2−m1). Charges z1, z2, z3, and z4 were calculated using each of the four pairs of consecutively charged masses (m1 and m2, m2 and m3, m3 and m4, m4 and m5), subtracted by the number of protons the peak has compared to m5, and averaged together and rounded to the nearest integer to calculate the lowest charge (z5). Charges z1-4 are recalculated based on charge z5 (z1=z5+4, z2=z5+3, etc.), uncharged masses are calculated from each of the five m/z's: uncharged mass=zx(observed m/z)−zx.
Peptide expression in 96-well plates. Plasmids were transformed into E. coli NEB Express using 15 μL of competent cells and 1 μL of each plasmid being transformed in a 96-well PCR plate (1402-9596, USA Scientific, FL, USA or 951020401, Eppendorf, N.Y., USA). Transformations were incubated on ice (20-30 min), heat shocked (40° C., 30 sec), and incubated on ice again (5 min). Cells were then transferred to a deep well 96-well plate (1896-2000, USA Scientific, FL, USA) with 100 μL of SOC media. After outgrowth (Multitron Pro, 1 hr, 37° C.) in an Infors HT Multitron Pro (Infors USA, MD, USA), 400 μL LB media was added with appropriate antibiotics (100 μg/ml carbenicillin and 50 μg/ml kanamycin) and incubated (Multitron Pro, 30° C., 900 r.p.m.) until all wells reached stationary phase (cultures were visibly saturated, 12-30 hours). Overnight cultures were diluted 1:100 into 1 mL LB or TB media (with same antibiotics as previous culture) in deep well plates. After a 3 hour incubation (Multitron Pro, 30° C., 900 r.p.m.), appropriate inducer was added (1 mM IPTG or 200 μM cumate) and cultures were incubated for 20 hours (Multitron Pro, 30° C., 900 r.p.m.). The 96-well plates were centrifuged (Legend XFR, 4,500 g, 4° C., 20 min) and media discarded. Pellets were either purified immediately or frozen at −20 C until purification.
Haloduracin production and purification. Haloduracin was produced following the 96-well expression protocol described above, with each sample being produced in two wells of 1 mL TB media to double the amount of product produced. Culture pellets were resuspended in 800 L lysis buffer, freeze-thawed (frozen at −80° C.; thawed in Multitron Pro at 37° C., 900 r.p.m), and centrifuged (Legend XFR, 4,500 g, 4° C., 30 min). Peptides were affinity purified using HIS MultiTrap TALON plates, using 500 μL water and two 500 μL lysis buffer washes for column equilibration (Legend XFR, 500 g, 4° C., 2 min), loading 600 μL of both matching sample's clarified lysates iteratively (load one, then centrifuge, then load the second, then centrifuge) (Legend XFR, 100 g, 4° C., 5 min), washing twice with 500 μL wash buffer, and eluting three times with 200 μL elution buffer to maximize titer. Purification was followed by solid-phase extraction (SPE) using Strata-XL microtiter plates (8E-S043-TGB, Phenomenex, CA, USA). Plates were conditioned with 1 mL methanol wash followed by 1 mL water wash. All 600 μL of TALON eluent was loaded, washed twice with 1 mL water, and then eluted twice with 500 μl 1:1 acetonitrile:water (supplemented with 0.1% formic acid). Plates with eluent were dried down at room temperature in a Savant Speedvac SPD2010 (Thermo Fisher Scientific, MA, USA), samples resuspended in 40 μL TE buffer (10 mM tris, 1 mM EDTA) with 20 μL 2 mg/mL TEV protease, and then incubated (stationary, 30° C., 8 hr). Cut fractions were desalted using a Strata-X SPE plate (8E-S100-TGB, Phenomenex, CA, USA) with same condition/wash/elution/drying steps as above. Dried down samples were resuspended in 50 μL 1:1 methanol:water.
Proteolytic cleavage and removal of SUMO. For purification of haloduracin for antimicrobial assays, TEV protease was purified as described previously 78 [Addgene #8827, concentrated to 2 mg/mL in TEV buffer (25 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM TCEP, 50% glycerol)]. For MS/MS analysis, TEV protease was prepared as a 50 mg/mL solution of 10% (w/w) TEV lyophilizate (Gene and Cell Technologies, CA, USA) in TEV Buffer.
The protein modification enzyme used with the sequences in Table 3 was PapB. The modification (mod) refers to the scaffold for the core peptide and correspond to L3 and L5 in
aPlpXY genes were synthesized/expressed as RBSPLpX + PlpX + RBSPlpY + PlpY.
GCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGAGAACGATCGTTGGCTGa
TTACCACCATCACCATCATCACGGGTCCCTGCAGGACTCAGAAGTCAATCAAGAAGCTA
AGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGAT
GGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGA
AGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTA
TTAGAATTCAAGCTGATCAGGCCCCTGAAGATTTGGACATGGAGGATAACGATATTATT
GAGGCTCACCGCGAACAGATTGGAGGTTGCATGTCATATTACGACTCCATTCCCACAAG
CGAGAACTTGTACTTTCAAGGGTGC
ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTG
TCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGA
GAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGG
AAAACTACCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCT
TTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAA
GGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGC
TGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATT
TTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAAT
GTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCA
CAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTG
GCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCG
AAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGG
GATTACACATGGCATGGATGAGCTCTACAAATAA
TTCAGCCAAAAAACTTAAGACCGCC
GGTCTTGTCCACTACCTTGCAGTAATGCGGTGGACAGGATCGGCGGTTTTCTTTTCTCT
TCTCAACCAATGgcggcgcgccatcgaatggcgcaaaacctttcgcggtatggcatgat
GTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAG
CCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACA
TTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTTATTGGCGTTGCC
ACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGC
CGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCT
GTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTAT
CCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTT
ATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAGGACG
GTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTA
GCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCT
CACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCG
GTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTT
GCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGT
TGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGATAGCTCATGTTATATCC
CGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGC
TTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCAGTCTCACT
GGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGG
CCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGATAA
TTGGTAACGAATCAGACAATTGACGGCTCGAGGGAGTAGCATAGGGTTTGCAGAATCCC
TGCTTCGTCCATTTGACAGGCACATTATGCATCGATGATAAGCTGTCAAACATGAGCAC
CTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGAC
GCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGAGGGTCTCAGTGCAACGA
TCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAA
AGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGA
AGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTT
GTACGACGGTATTAGAATTCAAGCTGATCAGGCCCCTGAAGATTTGGACATGGAGGATA
ACGATATTATTGAGGCTCACCGCGAACAGATTGGAGGTTGCATGTCATATTACGACTCC
ATTCCCACAAGCGAGAACTTGTACTTTCAAGGGTGC
ATGAGCAAAGGAGAAGAACTTTT
CACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTT
CTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATT
TGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGG
TGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTG
CCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTAC
AAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAA
GGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTA
ACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTC
AAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAA
TACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAAT
CTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTA
ACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA
TTCAGCCAAAAAAC
TTAAGACCGCCGGTCTTGTCCACTACCTTGCAGTAATGCGGTGGACAGGATCGGCGGTT
TTCTTTTCTCTTCTCAACAAGTGAGACCATGGgcggcgcgccatcgaatggcgcaaaac
AACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCC
CGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGC
GATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGT
CGTTGCTTATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTC
GCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGA
ACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCA
GTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCC
TGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTAT
TATTTTCTCCCATGAGGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTC
ACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTG
GCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGG
CGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCG
TTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATT
ACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGA
AGATAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGG
GGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAAT
CAGCTGTTGCCAGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAAC
CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGAC
TGGAAAGCGGGCAGTGATAA
TTGGTAACGAATCAGACAATTGACGGCTCGAGGGAGTAG
CATAGGGTTTGCAGAATCCCTGCTTCGTCCATTTGACAGGCACATTATGCATCGATGAT
AAGCTGTCAAACATGAGCACGCTTACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGG
CAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGC
CCTAACGGGGGGCCTTTTTTTGTTTCTGGTCTCCCAAGCGCTTAACGATCGTTGGCTGa
TGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAG
GTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCT
GTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTA
TCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTAC
AGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAG
TTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGA
TGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCA
CGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAA
GATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCC
TGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCA
ACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACAT
GGCATGGATGAGCTCTACAAATAA
TGAAGAGCGCAGAGGTGGTTGTGTTGCGAAAAAAA
AAAAAAACACCCTAACGGGTGTTTTTTTTTTTTTGGTGTCCCCACGTGTGGCGCTGGAG
CAGAACGTGCAATGGAAACCCAGGGTAAACTGATTGCAGCAGCACTGGGTGTTCTGCGT
GAAAAAGGTTATGCAGGTTTTCGTATTGCAGATGTTCCGGGTGCAGCCGGTGTTAGCCG
TGGTGCACAGAGCCATCATTTTCCGACCAAACTGGAACTGCTGCTGGCAACCTTTGAAT
GGCTGTATGAGCAGATTACCGAACGTAGCCGTGCACGTCTGGCAAAACTGAAACCGGAA
GATGATGTTATTCAGCAGATGCTGGATGATGCAGCAGATTTTTTTCTGGATGATGATTT
TAGCATCGGCCTGGATCTGATTGTTGCAGCAGATCGTGATCCGGCACTGCGTGAAGGTA
TTCTGCGTACCGTTGAACGTAATCGTTTTGTTGTTGAAGATATGTGGCTGGGTGTGCTG
GTGAGCCGTGGTCTGAGCCGTGATGATGCCGAAGATATTCTGTGGCTGATTTTTAACAG
CGTTCGTGGTCTGACAGTTCGTAGCCTGTGGCAGAAAGATAAAGAACGTTTTGAACGTG
TGCGTAATAGCACCCTGGAAATTGCACGTGAACGTTATGCAAAATTCAAACGTTGATAA
CAGAATCCCTGCTTCGTCCATTTGACAGGCACATTATGCATCGATGATAAGCTGTCAAA
CATGAGCAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGC
GGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCG
GGCTCATGAGCAAATATTTTATCTGAGGTGCTTCCTCGCTCACTGACTCGCTGCACGAG
TTCTTCCAACTGATCTGTGCGCGAGGCCAAGCGATCTTCTTCTTGTCCAAGATAAGCCT
GTCTAGCTTCAAGTATGACGGGCTGATACTGGGCCGGCAGGCGCTCCATTGCCCAGTCG
GCAGCGACATCCTTCGGCGCGATTTTGCCGGTTACTGCGCTGTACCAAATGCGGGACAA
CGTAAGCACTACATTTCGCTCATCGCCAGCCCAGTCGGGCGGCGAGTTCCATAGCGTTA
AGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAAAGAGTTCCTCC
GCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTTTGTCAGCAAGATAGCCAG
ATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCATTGCGCTGCCATT
CTCCAAATTGCAGTTCGCGCTTAGCTGGATAACGCCACGGAATGATGTCGTCGTGCACA
ACAATGGTGACTTCTACAGCGCGGAGAATCTCGCTCTCTCCAGGGGAAGCCGAAGTTTC
CAAAAGGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAA
CCAGCAAATCAATATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAA
TGTACGGCCAGCAACGTCGGTTCGAGATGGCGCTCGATGACGCCAACTACCTCTGATAG
TTGAGTCGATACTTCGGCGATCACCGCTTCCCTCATTTTAGCTTCCTTAGCTCCTGAAA
ATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGCGCGAAG
TCAAGGTGCGTACCTTGACTGATGAGTCCGAAAGGACGAAACACCCCTCTACAAATAAT
CTGCAAATACAACCACATTTCCATGTAGAGGTCATTGAACCAAAGCAAGTCTACTTGTT
GGGTGAACAAGCTAATCATGCATTGACAGGCCAATTATACTGCCAAATTTTGCCATTGT
TAAACGGACAATACACATTGGAACAAATCGTTGAAAAACTAGACGGAGAAGTACCACCT
GAATACATTGATTATGTGCTGGAGAGACTAGCTGAGAAGGGCTATCTGACTGAAGCAGC
ACCTGAATTATCTAGTGAAGTGGCCGCTTTCTGGTCTGAGCTGGGGATTGCACCTCCTG
TCGCGGCCGAAGCATTACGTCAACCTGTGACTTTAACACCTGTTGGAAACATCAGCGAA
GTAACAGTAGCAGCCTTAACCACAGCCCTACGTGATATCGGTATTTCCGTTCAAACACC
TACAGAAGCTGGATCGCCAACTGCATTGAACGTTGTACTTACCGATGATTATCTCCAAC
CAGAACTCGCTAAGATCAATAAGCAAGCCTTAGAAAGTCAACAAACTTGGCTACTTGTC
AAACCAGTTGGCTCCGTGTTATGGTTGGGTCCGGTATTCGTGCCAGGAAAAACAGGTTG
CTGGGATTGTTTGGCTCACAGATTAAGGGGGAATAGAGAGGTAGAGGCCTCTGTATTGA
GACAAAAACAAGCTCAACAACAACGTAATGGACAAAGCGGGTCTGTAATAGGATGCCTT
CCCACGGCTAGAGCGACACTGCCCTCAACACTCCAAACTGGGCTGCAGTTCGCTGCTAC
CGAAATTGCTAAATGGATAGTTAAGTATCATGTTAATGCCACAGCGCCTGGCACCGTAT
TCTTCCCTACATTGGATGGTAAGATAATTACGCTAAATCACTCCATACTGGATTTGAAG
TCACATATTCTGATCAAGCGTTCTCAATGTCCCACCTGTGGTGACCCAAAAATCTTACA
GCACCGTGGTTTCGAACCTTTAAAACTTGAGTCAAGGCCTAAACAGTTCACCTCAGACG
GCGGACATCGTGGTACTACCCCTGAACAAACTGTCCAGAAATATCAACATTTAATCTCG
CCTGTTACCGGTGTAGTTACTGAATTGGTCAGGATAACTGATCCGGCCAATCCACTAGT
TCACACATATAGAGCTGGTCATAGCTTCGGGAGCGCTACATCGCTGAGAGGGCTGCGTA
ATACCTTAAAGCATAAGAGTTCAGGTAAGGGTAAGACTGATTCTCAAAGTAAAGCCTCG
GGCCTGTGTGAGGCGGTAGAACGTTACTCAGGAATCTTTCAAGGTGACGAACCGAGAAA
ACGCGCCACATTGGCTGAATTGGGAGATTTGGCAATTCACCCTGAGCAATGCTTGTGTT
TTTCCGACGGTCAGTACGCTAATAGAGAAACTTTAAACGAACAGGCAACGGTGGCACAT
GATTGGATACCTCAACGTTTTGATGCATCACAAGCTATTGAATGGACTCCAGTCTGGTC
CCTAACTGAACAGACCCATAAATATTTGCCCACCGCATTGTGTTACTACCATTATCCTC
TACCCCCAGAACACAGATTCGCACGTGGAGATTCGAATGGTAATGCTGCCGGAAATACG
TTGGAAGAGGCTATACTCCAAGGCTTCATGGAATTAGTCGAGAGAGATGGTGTGGCTTT
ATGGTGGTATAACAGGCTACGCAGACCCGCTGTAGACTTAGGCTCATTTAACGAGCCAT
ACTTCGTTCAGTTGCAACAATTCTACAGAGAAAACGATAGAGATTTGTGGGTTTTGGAC
TTGACAGCTGATTTAGGTATCCCGGCTTTCGCGGGCGTTTCTAATAGAAAAACTGGTAG
TTCGGAGAGGTTGATATTAGGATTCGGTGCACACCTCGATCCTACTATTGCAATTCTGA
GAGCAGTTACAGAAGTTAACCAGATTGGCCTTGAATTAGATAAAGTTCCAGACGAGAAC
CTTAAGAGCGACGCAACAGATTGGCTAATTACTGAAAAATTAGCTGACCACCCTTATTT
GTTACCAGATACAACTCAACCTCTAAAAACTGCTCAAGATTATCCTAAAAGGTGGTCTG
ACGATATATACACGGACGTAATGACTTGCGTTAATATTGCTCAACAAGCAGGACTTGAA
ACTCTAGTTATTGATCAAACACGTCCGGACATTGGTTTGAATGTTGTTAAGGTGACAGT
CCCGGGGATGAGGCACTTTTGGTCAAGATTTGGAGAGGGGAGGCTTTATGACGTGCCCG
TCAAATTAGGTTGGCTTGACGAACCTTTGACCGAAGCGCAAATGAACCCCACGCCGATG
CCTTTTTAATAATGAAGAGCTAAGCGTTGAACGCTACACGGACTCTAACTAAAAAGGCC
TCCCAAATCGGGGGGCCTTTTTTATTGATAACAAAACGGCATGCGCATGGACGACTACG
TCGCTGAAATGCGACGAAACTTATGACCTCTACAAATAATTTTGTTTAAGAGCCACCAG
TTATAAGGAGAACCTACCG
ATGACCAAAAAGTATCGGCGTGTATCCTACGCAGTGTGGG
AAATCACCCTGAAATGCAATCTGGCATGCTCTCATTGTGGCAGCCGCGCCGGCCAAGCC
CGTACGAAAGAGCTGAGTACCGAAGAAGCGTTCAACCTGGTCCGCCAGCTGGCCGACGT
GGGCATTAAGGAAGTCACCCTGATCGGTGGTGAAGCCTTTATGCGTTCGGATTGGCTGG
AAATCGCGAAAGCCGTCACTGAAGCCGGCATGATCTGTGGCATGACCACAGGGGGCTTC
GGGGTCAGTCTGGAAACGGCGCGTAAAATGAAAGAAGCGGGCATTAAAACGGTGAGCGT
TAGCATTGACGGTGGTATTCCTGAAACCCACGACCGCCAGCGCGGTAAAAAGGGTGCGT
GGCATAGTGCATTCCGGACTATGAGCCATCTGAAAGAAGTCGGGATCTACTTCGGTTGC
AACACTCAAATCAATCGTTTATCGGCGTCAGAATTCCCGATTATCTATGAACGTATTCG
CGATGCTGGGGCACGTGCGTGGCAAATTCAGCTGACGGTTCCGATGGGCAACGCCGCGG
ATAACGCAGATATGCTGCTGCAACCGTATGAATTGCTCGACATCTATCCGATGTTAGCC
CGCGTTGCCAAACGTGCGAAACAGGAAGGCGTGCGTATTCAGGCAGGTAACAACATCGG
GTACTATGGACCGTATGAGCGTCTGCTGCGTGGCAGCGACGAATGGACGTTTTGGCAAG
GATGTGGTGCGGGCCTTAACACCCTCGGCATCGAAGCCGACGGCAAAATCAAAGGCTGT
CCATCCCTGCCGACCGCCGCGTACACCGGCGGTAACATTCGCGATCGCCCGCTGCGGGA
AATCGTCGAACAGACCGAAGAACTGAAATTTAACTTAAAAGCTGGTACAGAACAAGGTA
CGGACCATATGTGGGGCTTTTGTAAAACCTGCGAATTCGCGGAACTCTGTCGCGGCGGA
TGCAGCTGGACTGCGCATGTGTTCTTTGACCGGCGCGGCAATAATCCGTACTGCCACCA
TCGGGCTCTGAAACAAGCCCAAAAAGACATTCGCGAACGCTTTTATTTAAAAGTGAAAG
CAAAGGGCAACCCGTTCGACAATGGTGAATTTGTTATCATTGAAGAACCTTTTAACGCT
CCGTTACCCGAGAATGACCTGCTGCACTTTAACAGTGATCACATTCAATGGCCAGAAAA
CTGGCAAAATAGTGAAAGCGCGTACGCATTGGCCAAGTAATAAATATAAAGTTAAGGAG
TTGCACATGAACAGTAATCAGATCCCTAACAAAGTTGCAACCGCGGCACAGAAATCTGA
CGACAGCAGCAGCGTATTACCGCGCCAGGGGTGGCAAGACAAACAAGCCTTTATTAAGG
CACTCATTAAAGCCAAACAGTCTCTCGAAATTGCCGAAATTAGCAACTTTTTAACCTAA
CGTCTTTTTTCGTTTTGGTCCCACGTGGCAAGCGCTTAACGATCGTTGGCTGaacaaac
ACAGTGATGTCGAAACCGCCTCTACAAATAATTTTGTTTAAGCTCTTCAAGAGCATTCC
ATAAGGAGAAATTTT
ATGACGAGAACCAACACCGGCTATCGTTATCGCGCGTTCGGCCT
GCGCATAGACTCAGATATTCCGCTGCCAGAATTAGGGGACGGTACGCGCCCTGATGGTG
ACGCGGATCTGACGGTCGTCCGGTGTGGGGAAGCGGAGCCGGAATGGGCTGAAGGTGGT
GGCGGGGGTCGTCTGTATGCCGCTGAAGGCATTGTATCTTTTCGCGTGCCGCAGACGGC
AGCGTTCCGTATTACTAATGGAAATCGCATCGAGGTGCATGCCTACTCGGGGGCTGATG
AGGATCGAATACGCCTGTACGTGTTAGGGACCTGTATGGGAGCGCTGTTACTGCAACGT
AGAATCTTACCGCTTCATGGTTCGGTCGTCGCCCGTGATGGTCGTGCGTATGCCATAGT
TGGCGAAAGCGGAGCGGGCAAATCCACGATGAGTGCAGCACTTCTCGAACGTGGATTCC
GCCTCGTTACGGATGACGTGGCCGCCATCGTGTTCGATGAGCGTGGGACCCCACTGGTT
ATGCCGGCTTATCCACAGCAAAAACTGTGGCAGGATTCCCTGGACCGTCTGCAAATTGC
GGGCTCGGGCCTTCGTCCGCTGTTCGAACGCGAAACGAAATACGCTGTACCCGCGGATG
GGGCATTCTGGCCCGAACCGGTTCCATTGGTGCACATTTACGAACTGGTTCATAGCGAT
GGTCAAACGCCTGAACTGCAGCCGATTGCCAAATTAGAGCGTTGCTATACCTTGTATCG
CCACACATTTCGTAGAAGCCTGATCGTCCCCAGCGGCTTAAGCGCCTGGCATTTTGAAA
CGGCAGTGAAACTTGCGGAGAAAACGGGGATGTACCGTCTTATGCGCCCGGCCAAAGTT
TTCGCGGCTCGCGAATCTGCTCGGCTGATTGAAACTCACGCCGATGGTGAAGTGTCACG
TTAATAATGAAGAGCGGATGAGCTCTACAAATAAGCAGAGGTGGTTGTGTTGCGAAAAA
aText formatting correspond to sequence features/components: promoters (lowercase),
terminators (UNDERLINED), and plasmid backbone and spacers (REGULAR ALL CAPS). Each backbone plasmid sequence except for the multi-enzyme backbone includes a coding sequence for GFP. The portion of the sequence that is double underlined can be replaced with a peptide coding sequence or an RBS + enzyme coding sequence, for example chosen from Table 9, to generate a plasmid encoding a sequence of interest (e.g., a peptide or enzyme).
bThe multi-enzyme backbone shown is the sequence of the 6048 plasmid. To generate an alternative multi-enzyme plasmid, the protein coding sequences (shown in bold) can be replaced with coding sequences for alternative enzymes (e.g., one chosen from Table 9)
TAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGCatcc
TCATATTACCACCATCACCATCATCACGACTATGATATTCCCACAAGCATGAAA
ATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGA
TTGGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTT
GAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGAT
GGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCT
GGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCG
TTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCT
GTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAA
ACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGC
GCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCT
GACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTG
GGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATT
AAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTT
AATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATC
GACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAA
CCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCG
AACAAAGAGCTGGCGAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGT
CTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTAC
GAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCACCATGGAAAACGCCCAG
AAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTG
CGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTG
AAAGACGCGCAGACTCGTATCACCAAGTCGTACTACCATCACCATCACCATCAC
GGCGGTAGTGGCGAAAACCTGTATTTTCAGGGTATGAACAAGAAGAACATTTTA
CCGCAGTTAGGACAACCAGTCATCCGCCTTACTGCCGGTCAACTGTCAAGCCAA
CTGGCGGAGCTTTCTGAGGAGGCTCTGGGAGGGGTCGATGCCTCGTACGCGGTG
TTCTGGCCGATCTGTAGCTATGACGACTAATAA
TTCAGCCAAAAAACTTAAGAC
CGCCGGTCTTGTCCACTACCTTGCAGTAATGCGGTGGACAGGATCGGCGGTTTT
CTTTTCTCTTCTCAACTGTACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGG
CAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGT
CTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGAGA
ATATTCCCACAAGCGAGAACTTGTACTTTCAAGGG
ATGAGCAAAGGAGAAGAAC
TTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGC
ACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCA
CCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTG
TCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGA
AACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCA
CTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTG
AAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAG
ATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAATGTAT
ACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCC
ACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTC
CAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAAT
CTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGT
TTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA
TTCA
GCCAAAAAACTTAAGACCGCCGGTCTTGTCCACTACCTTGCAGTAATGCGGTGG
ACAGGATCGGCGGTTTTCTTTTCTCTTCTCAACCAATGgcggcgcgccatcgaa
GTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCG
AAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAAC
CGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTTATTGGCGTTGCCACC
TCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGC
GCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTC
GAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTG
ATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGC
ACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGT
ATTATTTTCTCCCATGAGGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCA
TTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCG
ATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATG
CAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAG
ATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCG
GATATCTCGGTAGTGGGATACGACGATACCGAAGATAGCTCATGTTATATCCCG
CCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGAC
CGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCA
GTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCT
CCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTG
GAAAGCGGGCAGTGATAA
TTGGTAACGAATCAGACAATTGACGGCTCGAGGGAG
TAGCATAGGGTTTGCAGAATCCCTGCTTCGTCCATTTGACAGGCACATTATGCA
TCGATGATAAGCTGTCAAACATGAGCACGCTTACTAGTAGCGGCCGCTGCAGTC
CGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAA
GATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG
ATGGCATATCCCAACGATCAACAAGGTAAAGCACTTCCTTTCTTTGCTCGTTTC
TTGTCCGTAAGCAAAGAGGAATCTTCCATCAAGTCTCCTTCCCCTGAGCCTACC
TACGGGGGCACCTTTAAATACCCTTCTGACTGGGAAGATTATTAATAA
ATGGGTCCGGTTGTTGTGTTCGATTGCATGACGGCCGACTTTCTGAACGACGAT
CCAAATAACGCGGAGTTGTCTGCCTTGGAAATGGAGGAGCTCGAGTCCTGGGGC
GCCTGGGACGGAGAGGCTACCAGCTAGTAA
ATGAGTAAGGAATTAGAAAAAGTTCTTGAATCCAGTTCAATGGCAAAGGGGGAC
GGCTGGAAGGTTATGGCTAAAGGTGACGGTTGGGAGTAATAA
ATGTGGAAGAAACCTGCTTTTATCGATTTACGTCTCGGTCTGGAAGTGACGCTG
TACATTTCTAACCGTTAATAA
ATGAAAAAAGCTGTCATTGTAGAAAACAAAGGTTGTGCAACATGCTCGATCGGA
GCCGCTTGTCTAGTGGACGGTCCTATCCCTGATTTTGAAATTGCCGGTGCAACA
GGTCTATTCGGTCTATGGGGATAA
ATGGATAACAAGGTTGCGAAGAATGTCGAAGTGAAGAAGGGCTCCATCAAGGCG
ACCTTCAAGGCTGCTGTTCTGAAGTCGAAGACGAAGGTCGACATCGGAGGTAGC
CGTCAGGGCTGCGTCGCTTAATAA
ATGTCAGAAGAACAACTCAAGGCATTCATTGCCAAGGTTCAAGCAGACACTTCA
CTGCAGGAACAGCTCAAAGTAGAAGGTGCTGATGTTGTTGCTATTGCTAAAGCC
TCAGGGTTCGCGATTACCACAGAGGACTTAAAAGCACATCAAGCCAACTCACAA
AAGAACCTGTCTGATGCTGAGCTGGAAGGTGTGGCTGGGCGAACCATTGGGGGA
ACCATTGTGTCGATAACCTGTGAGACTTGCGATCTGCTTGTGGGGAAAATGTGC
TGATAA
ATGGACCTGAATGATCTGCCGATGGATGTTTTTGAACTGGCAGATAGCGGTGTT
GCAGTTGAAAGCCTGACCGCAGGTCATGGTATGACCGAAGTTGGTGCAAGCTGT
AATTGCTTTTGTTATATTTGTTGTAGCTGCAGCAGCGCCTAATAA
ATGGAGCGCGAAATCGTGTGGACAGAAATTGAGGAGTCGGATTTAGCCGCCGTC
GTGTCGGCATCTAATGTCAAGGATGGTCCAACCGTTAGCTCAAGTAATGTAAAG
GACCGCTAATAA
CTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGAGA
TGCAGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCA
AGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCT
TCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAA
GACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTC
AAGCTGATCAGGCCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGG
CTCACCGCGAACAGATTGGAGGTCATCACCATCACCACCATGGATATGATATTA
GCACAGGTATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTG
TTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTG
AAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAA
AACTACCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAAT
GCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCA
TGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCT
ACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCG
AGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCG
AGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATG
GAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAAC
TAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTAC
CAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAA
AGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATG
GCATGGATGAGCTCTACAAATAA
TTCAGCCAAAAAACTTAAGACCGCCGGTCTT
GTCCACTACCTTGCAGTAATGCGGTGGACAGGATCGGCGGTTTTCTTTTCTCTT
CTCAACCAATGgcggcgcgccatcgaatggcgcaaaacctttcgcggtatggca
TTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTG
GTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCG
ATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAA
CAGTCGTTGCTTATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCG
CAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTG
GTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAAT
CTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAG
GATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGAT
GTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAGGACGGTACG
CGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTA
GCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAA
TATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGT
GCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCC
ACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATT
ACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGAT
ACCGAAGATAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTT
CGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAG
GCGGTGAAGGGCAATCAGCTGTTGCCAGTCTCACTGGTGAAAAGAAAAACCACC
CTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGATAATTGGTAACG
AATCAGACAATTGACGGCTCGAGGGAGTAGCATAGGGTTTGCAGAATCCCTGCT
TCGTCCATTTGACAGGCACATTATGCATCGATGATAAGCTGTCAAACATGAGCA
CCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCT
ATGAACAAGAAGAACATTTTACCGCAGTTAGGACAACCAGTCATCCGCCTTACT
GCCGGTCAACTGTCAAGCCAACTGGCGGAGCTTTCTGAGGAGGCTCTGGGAGGG
GTCGATGCCTCGTACGCGGTGTTCTGGCCGATCTGTAGCTATGACGACTAATAA
ATGGCATATCCCAACGATCAACAAGGTAAAGCACTTCCTTTCTTTGCTCGTTTC
TTGTCCGTAAGCAAAGAGGAATCTTCCATCAAGTCTCCTTCCCCTGAGCCTACC
TACGGGGGCACCTTTAAATACCCTTCTGACTGGGAAGATTATTAATAA
ATGAAAAAAGCTGTCATTGTAGAAAACAAAGGTTGTGCAACATGCTCGATCGGA
GCCGCTTGTCTAGTGGACGGTCCTATCCCTGATTTTGAAATTGCCGGTGCAACA
GGTCTATTCGGTCTATGGGGATAA
ATGTGGAAGAAACCTGCTTTTATCGATTTACGTCTCGGTCTGGAAGTGACGCTG
TACATTTCTAACCGTTAATAA
ATGAGTAAGGAATTAGAAAAAGTTCTTGAATCCAGTTCAATGGCAAAGGGGGAC
GGCTGGAAGGTTATGGCTAAAGGTGACGGTTGGGAGTAATAA
ATGGGTCCGGTTGTTGTGTTCGATTGCATGACGGCCGACTTTCTGAACGACGAT
CCAAATAACGCGGAGTTGTCTGCCTTGGAAATGGAGGAGCTCGAGTCCTGGGGC
GCCTGGGACGGAGAGGCTACCAGCTAGTAA
ATGGATAACAAGGTTGCGAAGAATGTCGAAGTGAAGAAGGGCTCCATCAAGGCG
ACCTTCAAGGCTGCTGTTCTGAAGTCGAAGACGAAGGTCGACATCGGAGGTAGC
CGTCAGGGCTGCGTCGCTTAATAA
ATGTCAGAAGAACAACTCAAGGCATTCATTGCCAAGGTTCAAGCAGACACTTCA
CTGCAGGAACAGCTCAAAGTAGAAGGTGCTGATGTTGTTGCTATTGCTAAAGCC
TCAGGGTTCGCGATTACCACAGAGGACTTAAAAGCACATCAAGCCAACTCACAA
AAGAACCTGTCTGATGCTGAGCTGGAAGGTGTGGCTGGGCGAACCATTGGGGGA
ACCATTGTGTCGATAACCTGTGAGACTTGCGATCTGCTTGTGGGGAAAATGTGC
TGATAA
ATGGACCTGAATGATCTGCCGATGGATGTTTTTGAACTGGCAGATAGCGGTGTT
GCAGTTGAAAGCCTGACCGCAGGTCATGGTATGACCGAAGTTGGTGCAAGCTGT
AATTGCTTTTGTTATATTTGTTGTAGCTGCAGCAGCGCCTAATAA
ATGGAGCGCGAAATCGTGTGGACAGAAATTGAGGAGTCGGATTTAGCCGCCGTC
GTGTCGGCATCTAATGTCAAGGATGGTCCAACCGTTAGCTCAAGTAATGTAAAG
GACCGCTAATAA
CTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGAGA
TGCAGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCA
AGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCT
TCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAA
GACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTC
AAGCTGATCAGGCCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGG
CTCACCGCGAACAGATTGGAGGTTGCATGTCATATTACGACTCCATTCCCACAA
GCGAGAACTTGTACTTTCAAGGGTGC
ATGAGCAAAGGAGAAGAACTTTTCACTG
GAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTT
CTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAAT
TTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTACTC
TGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATG
ACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTT
TCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATA
CCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACA
TTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGG
CAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTG
AAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCG
ATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTT
CGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTG
CTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA
TTCAGCCAAAAAA
CTTAAGACCGCCGGTCTTGTCCACTACCTTGCAGTAATGCGGTGGACAGGATCG
GCGGTTTTCTTTTCTCTTCTCAACCAATGgcggcgcgccatcgaatggcgcaaa
CAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGG
GAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCA
CAACAACTGGCGGGCAAACAGTCGTTGCTTATTGGCGTTGCCACCTCCAGTCTG
GCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAA
CTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGT
AAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAAC
TATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTT
CCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTC
TCCCATGAGGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCAC
CAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGT
CTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAA
CGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTG
AATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTG
GGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCG
GTAGTGGGATACGACGATACCGAAGATAGCTCATGTTATATCCCGCCGTTAACC
ACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTG
CAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCAGTCTCACTG
GTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG
TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGG
CAGTGATAA
TTGGTAACGAATCAGACAATTGACGGCTCGAGGGAGTAGCATAGG
GTTTGCAGAATCCCTGCTTCGTCCATTTGACAGGCACATTATGCATCGATGATA
AGCTGTCAAACATGAGCACGCTTACTAGTAGCGGCCGCTGCAGTCCGGCAAAAA
AGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTC
ATGGCACTTCCTTTCTTTGCTCGTTTCTTGTCCGTAAGCAAAGAGGAATCTTCC
ATCAAGTCTCCTTCCCCTGAGCCTACCTACGGGGGCACCTTTAAATACCCTTCT
GACTGGGAAGATTATTAATAA
ATGTCAGAAGAACAACTCAAGGCATTCATTGCCAAGGTTCAAGCAGACACTTCA
CTGCAGGAACAGCTCAAAGTAGAAGGTGCTGATGTTGTTGCTATTGCTAAAGCC
TCAGGGTTCGCGATTACCACAGAGGACCTCAATTCGCATCGCCAAAATCTGTCT
GATGATGAGCTGGAGGGAGTCGCGGGAGGCTTTTTCTGCGTACAGGGTACGGCC
AACCGTTTCACTATCAACGTTTGCTGATAA
ATGATTAAATTTTCTACATTGTCTCAGCGCATCAGCGCCATCACGGAAGAAAAC
GCCATGTACACTAAGGGTCAAGTGATCGTATTGAGCTGATAA
ATGAAAAAAGCTGTCATTGTAGAAAACAAAGGTTGTGCAACATGCTCGATCGGA
GCCGCTTGTCTAGTGGACGGTCCTATCCCTGATTTTGAAATTGCCGGTGCAACA
GGTCTATTCGGTCTATGGGGATAA
CTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGAGG
TCACGGGTCCCTGCAGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAA
GCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTC
AGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGC
GTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGG
TATTAGAATTCAAGCTGATCAGGCCCCTGAAGATTTGGACATGGAGGATAACGA
TATTATTGAGGCTCACCGCGAACAGATTGGAGGTTGCATGTCATATTACGACTC
CATTCCCACAAGCGAGAACTTGTACTTTCAAGGGTGC
ATGAGCAAAGGAGAAGA
ACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGG
GCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACT
CACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACT
TGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACAT
GAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACG
CACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTT
TGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGA
AGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAATGT
ATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCG
CCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATAC
TCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACA
ATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGA
GTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA
TT
CAGCCAAAAAACTTAAGACCGCCGGTCTTGTCCACTACCTTGCAGTAATGCGGT
GGACAGGATCGGCGGTTTTCTTTTCTCTTCTCAACAAGTGAGACCATGGgcggc
AGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCC
ACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATT
ACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTTATTG
GCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGA
TTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAAC
GAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCG
TCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGG
AAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACAC
CCATCAACAGTATTATTTTCTCCCATGAGGACGGTACGCGACTGGGCGTGGAGC
ATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTT
CTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATC
AAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTC
AACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTG
CCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGC
GCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGATAGCTCAT
GTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAA
CCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATC
AGCTGTTGCCAGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGC
AAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGG
TTTCCCGACTGGAAAGCGGGCAGTGATAA
TTGGTAACGAATCAGACAATTGACG
GCTCGAGGGAGTAGCATAGGGTTTGCAGAATCCCTGCTTCGTCCATTTGACAGG
CACATTATGCATCGATGATAAGCTGTCAAACATGAGCACGCTTACTAGTAGCGG
AAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCT
AGCAAGAAAGAATGGCAAGAGCCCACGATCGAAGTGCTCGATATTAATCAGACT
ATGGCGGGTAAGGGCTGGAAACAGATAGACTGGGTGAGCGACCATGATGCTGAC
TTACACAATCCGTCTTAATAA
CTGAAAATCCGCAAGGTGAAAATTGTCAGAGCGCAGAACGGCCACTACACGAAC
TAATAA
GAAGCAGTTAAAGAGAAGAACGATCTGTTCAACCTGGATGTTAAAGTCAACGCA
AAAGAAAGTAACGATAGTGGCGCAGAACCACGCATAGCGTCGAAATTTATTTGC
ACACCAGGCTGCGCGAAAACGGGTTCGTTTAACAGCTATTGTTGTTAATAA
ACGAACTTGCTGAAAGAATGGAAAATGCCCCTGGAACGTACGCATAATAACTCC
AACCCGGCGGGAGACATTTTTCAGGAACTGGAAGATCAAGACATACTCGCCGGT
GTGAATGGAGCAGAAAACTTATACTTTCAGGGTTGTGCGTGGTATAACATTAGC
TGCCGTCTGGGCAACAAAGGAGCCTACTGCACCCTTACAGTTGAGTGCATGCCC
TCCTGTAACTGATAA
GTGAATTCCAAAGACCTGAGAAATCCAGAATTTCGCAAAGCTCAGGGTCTGCAG
TTTGTAGATGAAGTTAATGAGAAGGAACTCTCGAGTTTAGCCGGCAGCGAGAAT
CTTTACTTTCAAGGCACGACGTGGCCATGTGCGACCGTCGGCGTTTCAGTTGCC
TTGTGCCCGACGACCAAATGCACTTCACAGTGCTGATAA
TTGAAACAGATCAATGTGATTGCTGGCGTAAAAGAGCCTATTCGCGCCTATGAG
AACTTGTATTTCCAGGGTTGTTCGGCTAATGACGCATGCTATTTTTGCGACACG
CGTGACAACTGCAAAGCCTGTGATGCCAGTGATTTTTGTATCAAAAGTGATACG
AACAAGAAGAACATTTTACCGCAGTTAGGACAACCAGTCATCCGCCTTACTGCC
GGTCAACTGTCAAGCCAACTGGCGGAGCTTTCTGAGGAGGCTCTGGGAGAGAAC
TTGTATTTCCAGGGTGTCGATGCCTCGACCTTGCCGGTTCCGACGTTGTGTAGC
TATGACGGGGTGGACGCTAGCACAGTCCCTACACTTTGTAGTTACGATGAC
ATGAGCAAAAATGAGAACAACAAGAAACAGCTGCGCGATCTTTTCATTGAAGAT
CTGGGCAAAGTTACTGGCGAGAACTTGTATTTCCAGGGTAAAGGTGGCCCGTAT
ACCACCTTAGCCATTGGCGAAGAAGATCCGATTACCACTTTGGCTATCGGAGAA
GAGGACCCTGATCCAACGACACTTGCCTTAGGTGAAGAGGACCCAACTACGCTT
GCAATCGGCGAAGAA
ATGCCAGCCGATATTCTGGAGACTCGTACCAGCGAAACGGAGGACTTACTGGAT
CTTGACCTGAGCATCGGTGTAGAAGAAATCACCGCAGGCCCGGCAGTGACTTCT
TGGTCACTGTGCACCCCTGGATGCACGAGTCCGGGCGGTGGCTCCAATTGTTCG
TTCTGTTGCTAATAA
ATGAGCATTGAGAATGCCAAGAGCTTTTATGAACGCGTCAGTACAGATAAGCAG
TTCCGCACTCAACTGGAAAATACGGCCAGTGCTGAAGAACGGCAGAAAATCATT
CAGGCAGCGGGCTTTGAGTTCACCAATCAGGAGTGGGAAATTGCAAAAGAACAG
ATTCTTGCGACAAGTGAAAGTAATAACGGTGAACTGTCCGAGGCCGAACTGACC
GCCGTCAGCGGTGGGGTTGACTTAAGCATTTTCGAGCTGCTGGACGAAGAACCT
TTATTCCCGATTCGTCCTTTGTACGGCCTGCCTATTTAATAA
ATGTCTATTGAGAGTGCAAAGGCTTTCTACCAGCGTATGACGGATGACGCATCT
TTTCGTACCCCTTTTGAAGCGGAACTGTCGAAAGAGGAGCGCCAACAATTAATC
AAAGATAGCGGATATGACTTTACTGCAGAAGAATGGCAACAGGCTATGACCGAG
ATCCAGGCGGCACGCTCAAACGAGGAACTGAATGAGGAAGAACTCGAGGCAATT
GCCGGGGGCGCTGTGGCCGCAATGTATGGTGTGGTTTTCCCATGGGACAACGAG
TTCCCGTGGCCCCGCTGGGGCGGTTAATAA
ATGAACCTGAACGATTTACCTATGGACGTCTTTGAAATGGCAGACAGCGGTATG
GAGGTGGAAAGCCTCACGGCTGGCCATGGCATGCCAGAAGTTGGAGCTAGTTGC
AACTGTGTGTGCGGGTTTTGCTGCAGCTGCAGTCCGAGCGCGTAATAA
ATGAATAAAAACGAAATCGAAACCCAGCCAGTTACGTGGCTGGAGGAAGTTTCT
GATCAGAATTTTGATGAGGATGTCTTTGGTGCGTGTAGCACAAACACCTTCTCG
CTGAGCGATTACTGGGGTAACAACGGTGCTTGGTGTACACTCACGCACGAATGT
ATGGCATGGTGCAAGTAATAA
ATGAAGGAAAAGAATATGAAGAAAAACGACACCATCGAACTTCAGCTTGGAAAA
TACCTGGAAGATGATATGATCGAACTGGCTGAAGGGGATGAGTCCCATGGGGGT
ACTACCCCGGCTACCCCTGCGATTTCTATCCTCAGCGCGTATATCAGCACCAAT
ACCTGCCCGACAACTAAGTGTACACGCGCGTGCTAATAA
ATGTCCGAACTGAGTATGGAGAAAGTGGTCGGCGAAACATTTGAGGATCTGAGC
ATCGCGGAAATGACGATGGTGCAGGGCAGCGGCGACATTAACGGCGAATTTACT
ACCTCGCCGGCATGTGTTTATTCCGTTATGGTTGTATCGAAAGCAAGCAGCGCT
AAATGTGCGGCCGGTGCATCGGCAGTCTCGGGAGCCATTCTGAGTGCGATTCGT
TGCTAATAA
ATGAGCGAATCCAACATGAAGAAGGTTGTTGGCGAAACCTTCGAAGATCTGAGC
ATCGCAGAAATGACGAAAGTTCAGGGCTCAGGGGACGTGATGCCGGAATCTACC
CCAATTTGTGCCGGCTTCGCAACCTTGATGAGTTCTATCGGTCTTGTTAAAACC
ATCAAAGGCAATGTCAAAAGTTTCTCCGTCTTAATTTAATAA
ATGACCAATGAAGAGATCATTGTCGCGTGGAAAAACCCTAAAGTCCGTGGCAAA
AATATGCCAAGTCACCCGAGCGGCGTGGGATTCCAAGAGCTTTCCATCAACGAG
ATGGCCCAAGTGACCGGCGGAGCAGTAGAACAGCGTGCAACACCAACCCTGGCA
ACCCCGCTGACCCCGCATACCCCGTACGCAACCTATGTGGTTAGCGGAGGCGTG
GTTAGCGCGATTTCTGGTATCTTCAGCAACAATAAAACGTGTCTGGGCTAATAA
ATGACCAATGAGGAAATTATCGTTGCGTGGAAAAACCCGAAGGTGCGCGGCAAA
AACATGCCTTCCCATCCGTCCGGTGTGGGCTTCCAGGAATTATCTATTAATGAA
ATGGCACAGGTGACTGGTGGCGCGGTTGAACAGCGCGCGACGCCGGCAACCCCA
GCAACACCATGGCTGATTAAAGCGTCTTATGTGGTGAGTGGGGCGGGAGTTTCT
TTTGTCGCAAGCTATATCACTGTAAACTAATAA
ATGACGGCGAGTATTCTTCAGTCTGTCGTTGATGCGGACTTTCGTGCGGCCCTG
ATTGAAAACCCAGCCGCATTCGGCGCGAGCACCGCAGTTTTGCCGACCCCAGTC
GAACAGCAGGATCAGGCATCACTGGATTTTTGGACAAAAGATATTGCTGCCACT
GAGGCGTTTGCTTGCAAACAGTCTTGCTCATTTGGGCCGTTCACCTTTGTGTGC
GACGGGAATACCAAATAATAA
ATGGACAAACGTGTGCGTTACGAAAAACCGAGCCTGGTGAAAGAGGGTACGTTT
CGCAAAACTACCGCTGGCCTGCGGCGTCTGTTCGCTGACCAGCTGGTTGGCCGC
CGTAACATTTAATAA
ATGGATTCACTGCTGTCAACAGAAACCGTCATTAGTGATGACGAACTGCTTCCG
ATTGAAGTTGGTGGTACCGCGGAATTGACAGAGGGGCAGGGCGGCGGTCAGTCC
GAGGATAAACGTCGCGCTTATAACTGCTAATAA
ATGGAATTAAAAGCGAGTGAATTTGGTGTAGTTTTGTCCGTTGATGCTCTTAAA
TTATCACGCCAGTCTCCATTAGGTGTTGGCATTGGTGGTGGTGGCGGCGGCGGC
GGCGGCGGCGGTAGCTGCGGTGGTCAAGGTGGCGGTTGTGGTGGTTGCAGCAAC
GGTTGTAGTGGTGGAAACGGTGGCAGCGGCGGAAGTGGTTCACATATCTAATAA
ATGAGCAAAAATGAGAACAACAAGAAACAGCTGCGCGATCTTTTCATTGAAGAT
CTGGGCAAAGTTACTGGCGGTAAAGGTGGCCCGTATACCACCTTAGCCATTGGC
GAAGAAGATCCGATTACCACTTTGGCTATCGGAGAAGAGGACCCTGATCCAACG
ACACTTGCCTTAGGTGAAGAGGACCCAACTACGCTTGCAATCGGCGAAGAATAA
TAA
ATGCCGGAAAATCGGCAGGAAGATCTCAACGCTCAGGCTGTACCATTCTTCGCG
CGTTTCTTGGAGGGTCAAAACTGCGAGGACCTTACTGATGAGGAATCGGAGGCG
GTTAGCGGTGGAAAACGCGGCCAAACCCGTAAATATCCAAGCGACTGCGAAGAT
GGGAATGGCGTGACCGGTAAACTGCGCGATGAAGATATTGCAGTGACCTTGAAG
TACCCATCCGACAATGAAGATAATGGCGGCGGTGAAATTGTGACTCTGAAGTTT
CCAAGTGATGATGATGATCAACCAGTAGGCTAATAA
ATGTTGAAACAGATCAATGTGATTGCTGGCGTAAAAGAGCCTATTCGCGCCTAT
GGTTGTTCGGCTAATGACGCATGCTATTTTTGCGACACGCGTGACAACTGCAAA
GCCTGTGATGCCAGTGATTTTTGTATCAAAAGTGATACGTAATAA
ATGTCGAGTAATATCCTCGAAAAAGTTAAGGAGTTTTTCGTCCGGCTGGTGAAG
GATGATGCGTTTCAAAGCCAGCTGCAGAACAACAGTATTGATGAAGTTCGAAAT
ATCCTGCAGGAGGCCGGGTACATATTCAGCAAAGAAGAATTCGAAACCGCAACC
ATTGAATTGCTGGATTTGAAGGAACGCGATGAATTCCACGAGCTGACAGAAGAG
GAGCTTGTCACCGCTGTTGGCGGTGTTACGGGCGGGAGTGGTATATATGGCCCG
ATTCAAGCTATGTACGGTGCCGTCGTAGGTGATCCAAAACCGGGTAAGGACTGG
GGGTGGCGCTTTCCGAGCCCGCTGCCAAAACCGAGTCCGATTCCGAGTCCGTGG
AAACCCCCGGTTGATGTCCAGCCTATGTATGGTGTGGTAGTGTCAAACGATAGT
TAATAA
AAAAAGCAATATAGCAAACCTAGCCTGGAGGTTCTGGACGTCCACCAGACCATG
GCTGGCCCGGGCACTAGTACGCCAGACGCGTTTCAGCCAGATCCAGATGAAGAT
GTTCACTATGATTCGTAATAA
CGCAAGAAAGAATGGCAGACACCAGAACTGGAAGTACTCGATGTACGCCTCACC
GCAGCGGGCCCGGGTAAAGCTAAACCGGATGCTGTGCAGCCAGACGAAGATGAA
ATAGTGCACTACTCATAATAA
AAGAAATTCTATGAAGCGCCAGCTCTCATCGAACGTGGCGCCTTTGCGGCTGCT
ACAGCGGGGTTTGGACGTCTGCTGGCGGATCAGCTGGTGGGACGCCTGATTCCG
TAATAA
ACTAAAGGCCTGGACAAAATGCTTTTAACCAAAAAGAAGAAGGATAGTATGGGT
CTGCTGAACGAAATCGACGTTACCACCCTGGATGAACAGTTAGGCGGTAAAATG
AGCAAAGCATGGTGCCGATCCATGGTGGTGTCCTGCGTGTATAACCTGGTTGAT
TTTTCGTCGTCGAGTGACGGGAAAAAGACATGTGCTCTGTACCGCAAATATTGT
TAATAA
AAAGATCTTCTGAAGGAACTGATGTATGAAGTAGACCTCGAAGAGATGGAGAAT
CTTCAGGGTAGCGGGTACTCAGCCGCCCAGTGTGCCTGGATGGCGCTGAGCTGC
GTCAATTACATCCCGGGAGTGGGATTCGGTTGTGGCGGCTACAGCGCATGTGAA
CTCTACAAGCGTTATTGTTAATAA
AACCACTCTAAGAAAAGTCCGGCAAAAGGGGCAGCGTCCCTGCAGCGTCCTGCT
GGGGCAAAAGGCCGCCCTGAACCTCTGGATCAACGCTTGTGGAAACACGTCGGT
GGTGGTGACTACCCACCCCCAGGAGCCAACCCAAAGCATGATCCACCACCCCGC
AATCCGGGCCACCATTAATAA
CAAGATCTGATTAATTACTTCCTGAATTATCCTGAGGCTCTGAAGAAACTCAAG
AATAAGGAAGCCTGCTTAATTGGGTTTGACGTCCAGGAAACCGAAACGATTATC
AAAGCCTATAACGATTACTACCGCGCTGATCCGATCACGCGTCAATGGGGTGAT
TAATAA
AAGAACCCGACGCTGTTGCCCAAACTGACCGCGCCGGTCGAACGTCCGGCCGTA
ACTTCGTCGGATTTAAAGCAAGCCTCAAGCGTCGATGCTGCATGGTTAAATGGC
GATAATAACTGGTCAACCCCATTCGCCGGTGTGAACGCGGCATGGTTAAATGGG
GACAACAACTGGTCCACGCCTTTTGCGGGCGTGAATGCTGCATGGCTTAATGGC
GACAATAACTGGAGCACTCCATTTGCCGCCGATGGCGCTGAGTAATAA
TATCGACCTTATATTGCCAAGTATGTCGAAGAACAAACTCTGCAGAATTCAACC
AACCTGGTATATGACGACATCACGCAGATCTCTTTTATCAATAAAGAAAAGAAC
GTGAAAAAAATTAATCTGGGTCCCGATACTACGATCGTGACTGAAACCATCGAG
AATGCGGACCCCGATGAGTATTTCTTATAATAA
TCTGGTCGCGGGCGCGATCCTGATGCTGCTGTACCTCCCTTGCCTCGTGTACCT
CGCACTACTAATCATGAGCCACGTACGGCGTCCCGAGAACCAAGAGCAGCTCCA
AGAACTGGACCTACACGTCCGCCTTCGTCGCGTCCATCTCCGTGTGGTCACTCT
CCTCAAACCCCTGGTGCAGGACGCAGTGGATGTCGTGTGGAGCGTCAAAAATCG
GCTGCGGCTTCGTCTGAGAAGGAAAAGACAATGGAGAACCAAGATTTGGAGTTA
TTAGCACGCCTGCATGCACTTCCTGAGACTGAACCGGTGGGCGTCGACGGATTA
CCCTATGGCGAGACTTGTGAGTGCGTCGGGTTACTTACGTTGTTGAACACCGTA
TGTATCGGCATTTCATGCGCTTAATAA
ATGAACAAGAAGAACATTTTACCGCAGTTAGGACAACCAGTCATCCGCCTTACT
GCCGGTCAACTGTCAAGCCAACTGGCGGAGCTTTCTGAGGAGGCTCTGGGAGTC
GATGCCTCGACCTTGCCGGTTCCGACGTTGTGTAGCTATGACGGGGTGGACGCT
AGCACAGTCCCTACACTTTGTAGTTACGATGAC
CTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGAGA
AAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTG
ATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAA
ACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGT
GGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATC
CGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATG
TACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTG
AAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTG
ATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACT
CACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACT
TCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATC
AACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACC
TGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGG
TCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCT
ACAAA
CGACTGGTTCCGCGTGGTAGCTATTACGACTCCATTCCCACAAGCGAGA
ACGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGC
CTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCA
AGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGAC
AGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAG
CTGATCAGGCCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTC
ACCGCGAACAGATTGGAGGCTCCATTACAAGCCACCATCACCATCATCACGGTT
AATACTTTCAGCCAAAAAACTTAAGACCGCCGGTCTTGTCCACTACCTTGCAGT
AATGCGGTGGACAGGATCGGCGGTTTTCTTTTCTCTTCTCAACAAGTGAGACCA
TGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCA
GGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGA
GCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTT
GCTTATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGT
CGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGAT
GGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGC
GCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCAT
TGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGA
CCAGACACCCATCAACAGTATTATTTTCTCCCATGAGGACGGTACGCGACTGGG
CGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCC
ATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCAC
TCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTC
CGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGAT
GCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTC
CGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGA
TAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCT
GGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAA
GGGCAATCAGCTGTTGCCAGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCC
CAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC
ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGATAA
TTGGTAACGAATCAGAC
AATTGACGGCTCGAGGGAGTAGCATAGGGTTTGCAGAATCCCTGCTTCGTCCAT
TTGACAGGCACATTATGCATCGATGATAAGCTGTCAAACATGAGCACGCTTACT
TTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACT
ATGGTGCGTTTCCTGGCTAAGCTGCTGCGTTCAACGATCCATGGCTCTAATGGC
GTGAGCCTCGACGCCGTCAGTTCCACGCATGGTACTCCGGGGTTTCAGACACCT
GATGCACGTGTTATTTCACGCTTTGGCTTTAAT
ATGGACAAACGTGTGCGTTACGAAAAACCGAGCCTGGTGAAAGAGGGTACGTTT
CGCAAAACTACCGCTGGCCTGCGGCGTCTGTTCGCTGACCAGCTGGTTGGCCGC
CGTAACATT
ATGGATTCACTGCTGTCAACAGAAACCGTCATTAGTGATGACGAACTGCTTCCG
ATTGAAGTTGGTGGTACCGCGGAATTGACAGAGGGGCAGGGCGGCGGTCAGTCC
GAGGATAAACGTCGCGCTTATAACTGC
CCGATCATTAGCGAAACGGTCCAGCCTAAAACGGCTGGCCTGATTGTTCTGGGC
AAGGCAAGCGCGGAAACGCGCGGATTGAGCCAAGGCGTGGAACCGGACATTGGT
CAGACGTACTTCGAAGAAAGCCGTATTAATCAGGAT
ACTCCCATTCAATCCAAATTCTGCCTCCTGCGCGTGGGCAGTGCCAAACGGCTG
ACGCAGTCATTCGACGTGGGAACTATTAAGGAAGGTTTAGTCAGCCAGTATTAT
TTTGCG
ACCCAGGTGAGCCCATCACCGCTGCGCCTGATTCGCGTCGGGAGAGCCTTGGAC
CTGACCCGCTCTATCGGGGATAGTGGGCTGCGTGAGTCCATGTCAAGCCAGACG
TACTGGCCC
AACACTTTAAAAACGCGTCTTATTCGCTTTGGGTCGGCTAAACGTCTGACGCGC
GCAGGTACGGGCGTGCTGTTACCTGAAACCAACCAGATTAAGCGCTACGATCCA
GCA
ACCACACCCAAATTTCGACTGATTCGGTTAGGTTCAGCTAAGCGATTGACCCGG
TCGGGAATCGGGGATGTGTTTCCGGAGCCAAACATGGTTCGCCGCTGGGAT
CAGCGTATAATAGATGAAACCACCGATGGTCTGATTGAACTGGGGGCGGCCAGC
GTACAGACACAGGGCGATGTTTTGTTTGCTCCGGAGCCTGGCGTGGGCCGACCT
CCAATGGGCCTTTCCGAAGAT
GAACGCATTGAAGATCATATTGATGATGAACTGATTGACCTGGGAGCTGCTTCG
GTTGAAACCCAGGGAGATGTGCTGAATGCACCGGAGCCTGGTATCGGTCGTGAA
CCGACAGGCTTGAGCCGCGAT
GAATTTGAAGGTATCCCATCACCGGATGCGCGTATTGATTTGGGTCTGGCGTCG
GAAGAAACCTGTGGTCAGATTTATGATCACCCGGAAGTAGGCATCGGTGCGTAC
GGGTGCGAGGGCCTGCAGCGT
ACCAAGAAAAACGCAACACAGGCCCCACGTTTAGTACGTGTAGGCGATGCTCAT
CGTTTGACCCAAGGTGCTTTCGTTGGACAGCCGGAAGCCGTAAATCCACTTGGA
CGTGAAATTCAAGGA
ACCAAAACACACAGACTGATCAGATTGGGCGACGCGCAACGCTTGACCCAGGGC
ACATTGACTCCGGGCTTACCGGAGGACTTTCTGCCGGGCCATTACATGCCGGGG
ACTTCACGTTTCCAACTCCTGCGCCTGGGAAAAGCCGATCGTTTGACGCGTGGC
GCGCTGGTCGGGCTCCTGATCGAAGATATTACTGTCGCTCGCTACGACCCTATG
TGAAGGCCTCCCTAACGGGGGGCCTTTTTTTGTTTCTGGTCTCCACGACGCTTA
TCCACGTTGAGATAATTGAGCCGAAGCAAGTGTATCTCCTGGGCGAACAGGGCA
ACCACGCTCTCACCGGGCAGCTCTACTGCCAAATTCTGCCTTTCTTAAACGGCG
AATACACCCGAGAACAAATTGTGGAAAAGCTCGATGGGCAGGTCCCGGAGGAAT
ATATCGACTTCGTACTCAGTCGTCTGGTGGAGAAGGGCTATCTAACTGAGGTGG
CTCCAGAACTATCCCTGGAAGTGGCAGCATTTTGGAGCGAATTGGGAATTGCCC
CTTCTGTAGTGGCAGAAGGGCTAAAGCAGCCAGTGACAGTGACAACGGCGGGCA
AGGGCATTAGGGAAGGGATAGTGGCTAACCTGGCAGCAGCGCTGGAGGAAGCTG
GCATTCAGGTGTCAGACCCAAGGGACCCAAAGGCCCCAAAGGCAGGGGATTCTA
CTGCCCAGCTTCAGGTGGTGCTGACCGATGACTATTTACAGCCGGAACTTGCAG
CGATCAACAAGGAAGCCTTAGAGCGCCAACAACCCTGGTTGCTGGTTAAGCCTG
TGGGCAGTATCCTCTGGTTGGGACCGTTGTTCGTTCCTGGGGAAACCGGATGTT
GGCACTGTCTTGCTCAACGATTGCAAGGCAACCGGGAAGTTGAAGCATCGGTAT
TGCAACAAAAGCGAGCGCTGCAGGAGCGCAACGGTCAAAATAAAAATGGTGCAG
TGAGTTGCTTGCCCACAGCACGGGCAACCCTACCTTCTACTCTACAAACAGGTT
TACAGTGGGCTGCCACTGAGATTGCTAAGTGGATGGTCAAGCGGCACCTCAATG
CCATAGCACCGGGAACGGCTCGTTTTCCCACTCTAGCTGGCAAGATATTTACAT
TCAACCAGACGACTCTGGAGTTGAAAGCTCATCCTCTGAGCCGACGACCGCAAT
GTCCCACCTGTGGCGATCGGGAAACTCTCCAACGGCGCGGGTTTGAACCACTGA
AGCTAGAGTCGCGCCCCAAACACTTCACCTCCGATGGCGGTCATCGCGCCATGA
CCCCAGAACAAACGGTGCAGAAGTACCAACACCTCATCGGGCCCATAACGGGGG
TAGTGACGGAACTGGTGCGAATTTCTGACCCTGCCAATCCCTTGGTGCATACCT
ACCGGGCTGGGCATAGCTTTGGCAGTGCTACGTCTCTGCGGGGGCTGCGCAATG
TCCTACGCCACAAGAGTTCTGGTAAAGGCAAGACCGATAGCCAATCTCGGGCCA
GCGGACTTTGCGAGGCGATCGAGCGCTATTCGGGCATTTTTCAGGGAGACGAAC
CCCGCAAGCGGGCAACTTTGGCTGAGTTGGGAGATTTGGCGATTCATCCAGAAC
AGTGTTTGCACTTTAGCGACAGGCAGTATGACAACCGGGAAAGCTCGAACGAGC
GAGCAACAGTGACTCACGACTGGATTCCCCAACGGTTCGATGCAAGTAAGGCTC
ACGACTGGACTCCCGTGTGGTCCCTAACGGAGCAAACCCATAAGTATCTGCCTA
CAGCCCTGTGCTATTACCGATACCCCTTCCCCCCAGAACACCGTTTCTGCCGTA
GTGACTCCAACGGAAACGCGGCGGGAAATACCCTGGAAGAGGCGATTTTGCAAG
GATTTATGGAACTGGTGGAACGGGATAGCGTGTGCCTGTGGTGGTACAATCGCG
TTAGCCGTCCGGCTGTGGATTTGAGTAGCTTTGACGAGCCTTATTTTTTGCAGT
TGCAGCAGTTCTATCAAACTCAAAATCGCGATCTGTGGGTACTGGATTTAACAG
CAGATTTGGGCATTCCGGCTTTTGTAGGGGTATCGAATCGGAAAGCCGGCAGCT
CGGAAAGAATAATTCTCGGCTTTGGAGCGCACCTGGACCCGACAGTTGCCATCC
TTCGCGCTCTTACGGAGGTCAACCAAATAGGCTTGGAATTGGATAAAGTTTCTG
ATGAGAGCCTCAAGAACGATGCCACGGATTGGTTAGTGAATGCTACATTGGCAG
CTAGTCCCTATCTCGTTGCCGATGCTAGCCAACCCCTCAAGACTGCGAAGGATT
ATCCCCGGCGTTGGAGTGACGATATTTACACCGATGTGATGACTTGTGTAGAAA
TAGCCAAGCAAGCAGGTCTAGAGACTTTGGTACTGGATCAGACCAGACCCGACA
TAGGTTTAAATGTGGTTAAAGTCATTGTGCCAGGAATGCGTTTTTGGTCGCGAT
TTGGCTCCGGTCGGCTCTATGACGTGCCAGTGAAGTTGGGATGGCGAGAGCAAC
CACTTGCTGAGGCACAAATGAACCCTACACCGATGCCATTTTAATAAGATACGA
TTTTTTTTTTTGGTCCTACTATCCTTAAACGCATATCGTGGTACAGGAGACCGT
CGACACATACAGAATAATTAATAAAATTAAAGCTTGTAGAAGCAATAATGATAT
TAATCAATGCTTATCTGATATGACTAAAATGGTACATTGTGAATATTATTTACT
CGCGATCATTTATCCTCATTCTATGGTTAAATCTGATATTTCAATCCTAGATAA
TTACCCTAAAAAATGGAGGCAATATTATGATGACGCTAATTTAATAAAATATGA
TCCTATAGTAGATTATTCTAACTCCAATCATTCACCAATTAATTGGAATATATT
TGAAAACAATGCTGTAAATAAAAAATCTCCAAATGTAATTAAAGAAGCGAAAAC
ATCAGGTCTTATCACTGGGTTTAGTTTCCCTATTCATACGGCTAACAATGGCTT
CGGAATGCTTAGTTTTGCACATTCAGAAAAAGACAACTATATAGATAGTTTATT
TTTACATGCGTGTATGAACATACCATTAATTGTTCCTTCTCTAGTTGATAATTA
TCGAAAAATAAATATAGCAAATAATAAATCAAACAACGATTTAACCAAAAGAGA
AAAAGAATGTTTAGCGTGGGCATGCGAAGGAAAAAGCTCTTGGGATATTTCAAA
AATATTAGGTTGCAGTGAGCGTACTGTCACTTTCCATTTAACCAATGCGCAAAT
GAAACTCAATACAACAAACCGCTGCCAAAGTATTTCTAAAGCAATTTTAACAGG
AGCAATTGATTGCCCATACTTTAAAAATTGATAAGGATCCTAATTGGTAACGAA
TCAGACAATTGACGGCTCGAGGGAGTAGCATAGGGTTTGCAGAATCCCTGCTTC
GTCCATTTGACAGGCACATTATGCATCGATGATAAGCTGTCAAACATGAGCAGA
TCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTG
CTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCG
GGCTCATGAGCAAATATTTTATCTGAGGTGCTTCCTCGCTCACTGACTCGCTGC
TGAAGGCCTCCCTAACGGGGGGCCTTTTTTTGTTTCTGGTCTCCCAAGCGCTTA
GGTCAACGCATGTGCTTTGCGTTCTGATGAGACAGTGATGTCGAAACCGCCTCT
CTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGG
CACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTC
ACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTT
GTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATG
AAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGC
ACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTT
GAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAA
GATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAATGTA
TACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGC
CACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACT
CCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAA
TCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAG
TTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA
GGA
CCTAACGGGTGTTTTTTTTTTTTTGGTGTCCCCACGTGTGGCGCTGGAGACCGT
CCAGGCAGAACGTGCAATGGAAACCCAGGGTAAACTGATTGCAGCAGCACTGGG
TGTTCTGCGTGAAAAAGGTTATGCAGGTTTTCGTATTGCAGATGTTCCGGGTGC
AGCCGGTGTTAGCCGTGGTGCACAGAGCCATCATTTTCCGACCAAACTGGAACT
GCTGCTGGCAACCTTTGAATGGCTGTATGAGCAGATTACCGAACGTAGCCGTGC
ACGTCTGGCAAAACTGAAACCGGAAGATGATGTTATTCAGCAGATGCTGGATGA
TGCAGCAGATTTTTTTCTGGATGATGATTTTAGCATCGGCCTGGATCTGATTGT
TGCAGCAGATCGTGATCCGGCACTGCGTGAAGGTATTCTGCGTACCGTTGAACG
TAATCGTTTTGTTGTTGAAGATATGTGGCTGGGTGTGCTGGTGAGCCGTGGTCT
GAGCCGTGATGATGCCGAAGATATTCTGTGGCTGATTTTTAACAGCGTTCGTGG
TCTGACAGTTCGTAGCCTGTGGCAGAAAGATAAAGAACGTTTTGAACGTGTGCG
TAATAGCACCCTGGAAATTGCACGTGAACGTTATGCAAAATTCAAACGTTGATA
AGGATCCTAATTGGTAACGAATCAGACAATTGACGGCTCGAGGGAGTAGCATAG
GGTTTGCAGAATCCCTGCTTCGTCCATTTGACAGGCACATTATGCATCGATGAT
AAGCTGTCAAACATGAGCAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCA
CCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGG
AAGATCGGGCTCGCCACTTCGGGCTCATGAGCAAATATTTTATCTGAGGTGCTT
AGCGAAGTGGTTTTTGGACCGAATCTTGAGAAGATTGTAGGAGAAAAGCGCCTC
AATTTTTGGCTCAAACTTATAGGTGAGGACCCGGAAAACCTGAAGGAGTTTCTC
TCGAGAAAGGGCAATTCTTTCGAAGAACAAACCTTACCGGAAAAGGAAGCTATC
GTTCCGAACCGCTTAGGTGAAGAGGCGCTGGAAAAAGTCCGCGAAGAACTTGAG
TTCCTCAATACTTACAGCACTAAACATGTGCGTCGCGTTAAAGAGTTGGGAGTG
CAGATCCCTTTCGAAGGGATTCTGCTGCCATTCATTAGCATGTATATCGAAAAA
TTTCAGCAGCAGCAACTTCGCAAAAAGATAGGGCCGATTCACGAAGAGATCTGG
ACGCAGATTGTTCAAGATATCACCTCCAAATTAAATGCGATTCTGCACCGTACC
CTGATCCTGGAACTGAATGTAGCTCGTGTTACCTCCCAACTTAAAGGTGATACT
CCGGAAGAAAGATTCGCCTACTACTCGAAAACCTATTTAGGCAAACGTGAAGTA
ACTCACCGTCTGTATAGCGAATATCCGGTGGTTCTGCGGTTGCTGTTCACCACC
ATTTCACACCACATTTCGTTCATTACGGAAATCCTTGAACGCGTTGCAAATGAC
CGTGAAGCCATTGAAACCGAATTTTCACCGTGTTCCCCGATTGGTACCCTCGCC
TCTCTCCACTTAAACTCGGGAGATGCTCACCATAAACAGCGTACTGTGACGATT
TTGGAATTCTCCTCCTCGCTGAAACTTGTCTACAAACCTCGCTCCCTCAAAGTT
GATGGGGTGTTCAACGGTTTACTCGCTTTCCTGAACGATAGAACGGGGGAAGTC
ATTAAGGACCAGTATTGCCCTAAGGTGTTACAGCGCGATGGCTACGGCTATGTG
GAATTTGTCACTCACCAGTCTTGTCAATCCCTTGAGGAAGTGTCAGACTTCTAC
GAGAGACTCGGCTCTCTGATGAGTCTGTCCTACGTACTGAATAGTTCTGACTTT
CATTTCGAGAACATTATAGCTCATGGTCCCTATCCTGTCCTGATCGATCTTGAA
ACCATCATTCATAATACAGCGGATAGCAGCGAGGAAACGTCTACCGCTATGGAT
CGCGCGTTCCGTATGTTGAACGATTCGGTGCTGTCCACTGGTATGCTTCCCTCC
TCTATTTATTATCGCGATCAGCCGAATATGAAGGGTCTGAACGTCGGAGGTGTG
AGCAAATCAGAAGGTCAGAAAACACCGTTCAAAGTTAATCAAATCGCCAATCGC
AACACCGATGAGATGCGTATCGAAAAAGATCACGTTACCCTGAGCAGCCAGAAA
AATCTGCCCATTTTTCAGTCTGCCGCAATGGAGAGCGTACATTTCTTAGATCAG
ATCCAGAAAGGCTTTACCTCCATGTATCAGTGGATCGAGAAGAACAAACAAGAA
TTTAAAGAACAGGTGCGTAAGTTTGAAGGTGTGCCGGTTCGTGCTGTTCTTCGG
AGCACGACTCGCTATACCGAACTGCTGAAATCTTCCTACCACCCTGACCTGCTC
CGCAGCGCGTTGGACCGTGAAGTACTGCTGAACCGTTTGACTGTTGACTCGGTA
ATGACCCCGTATCTCAAAGAGATTATTCCACTCGAGGTGGAAGATCTGCTGAAC
GGTGACGTGCCATACTTCTACACCCTGCCGGAAGAACGCGCCCTGTATCAGGAA
GCGTCTGCGATCAATAGTACGTTCTTTACCACTTCGATTTTCCATAAGATTGAC
CAGAAAATCGATAAGCTGGGTATCGAGGACCATACCCAGCAAATGAAGATCTTA
CACATGAGTATGCTTGCCTCTAACGCTAACCATTACGCCGATGTTGCCGACTTG
GATATTCAGAAAGGACACACCATTAAAAACGAACAGTACGTTGAGATGGCCAAA
GACATCGGTGATTACCTGATGGAGTTATCGGTCGAGGGTGAAAATCAAGGGGAA
CCAGATCTGTGTTGGATTTCGACCGTCCTGGAAGGGAGCTCTGAAATCATTTGG
GACATCAGCCCAGTGGGCGAAGATTTATACAACGGCAGCGCTGGCGTCGCTCTC
TTTTATGCGTACCTGTTCAAAATTACAGGTGAAAAGCGTTACCAAGAGATCGCA
TACAAAGCCCTGGTTCCGGTTCGCCGCAGTGTGGCCCAATTCCAGCACCATCCG
AATTGGAGCATTGGTGCGTTTAACGGAGCGTCAGGCTATCTGTACGCGATGGGT
ACGATAGCGGCCCTGTTTAATGATGAACGTTTGAAGCATGAAGTAACCCGCAGC
ATTCCGCACATTGAACCGATGATCCACGAGGATAAGATCTATGATTTCATTGGC
GGTTCCGCAGGGGCGCTGAAGGTGTTCCTGAGCCTGTCGGGGCTGTTTGACGAG
CCGAAGTTTTTGGAACTTGCCATTGCATGCAGCGAACATCTGATGAAAAACGCC
ATTAAAACGGATCAAGGTATCGGCTGGAAACCACCGTGGGAGGTCACCCCACTG
ACCGGTTTCAGCCATGGGGTTAGCGGCGTCATGGCATCCTTCATCGAACTGTAC
CAGCAAACCGGTGATGAGCGCTTGCTCAGTTACATTGATCAGAGTTTAGCCTAT
GAACGTTCCTTCTTCAGCGAACAAGAGGAGAACTGGCTGACTCCGAACAAAGAA
ACACCCGTGGTAGCTTGGTGCCACGGCGCGCCGGGAATTTTGGTATCACGACTG
CTTCTGAAGAAATGCGGCTATTTGGATGAAAAAGTCGAAAAAGAAATTGAGGTG
GCATTATCCACAACTATCCGTAAAGGCCTTGGTAACAATCGCAGTCTTTGCCAT
GGTGATTTCGGCCAGCTGGAAATTCTTCGCTTTGCGGCGGAAGTGTTAGGCGAT
AGCTATCTCCAGGAAGTTGTCAACAATCTGTCCGGCGAGTTGTATAATCTTTTC
AAAACGGAGGGATATCAGAGCGGAACCAGCCGCGGTACTGAATCCGTGGGCCTG
ATGGTAGGTCTGTCCGGGTTTGGGTATGGTTTACTTTCAGCGGCATATCCATCT
GCTGTCCCCTCAATCTTAACATTGGATGGTGAGATCCAGAAGTACCGGGAGCCT
CATGAAGCCTGA
TCAGTGCCGACGACGCTGCCGCATACTAACGACACCGATTGGCTCGAGCAATTA
CATGACATTTTGTCCATTCCTGTTACGGAAGAAATCCAGAAATATTTCCACGCC
GAAAATGATCTGTTCTCGTTTTTCTATACACCGTTCCTGCAGTTTACGTACCAG
AGCATGTCGGACTACTTTATGACCTTCAAGACCGATATGGCCCTGATCGAAAGA
CAGAGCCTCCTGCAAAGCACGCTGACCGCGGTACATCACCGACTCTTCCACTTA
ACGCATCGCACCCTTATTAGTGAAATGCATATTGATAAACTTACCGTTGGCCTG
AATGGCTCTACGCCGCACGAGCGCTACATGGATTTCAACCACAAATTCAACAAA
ACCTCGAAGTCGAAGAACCTGTTTAACATCTACCCAATTTTGGGAAAATTGGTC
GTTAACGAAACTCTGCGCACTATTAACTTCGTCAAGAAAATCATTCAGCACTAC
ATGAAGGACTACCTGCTCCTGTCGGACTTCTTCAAAGAGAAGGACTTGCGTCTT
ACCAACCTGCAATTAGGCGTGGGGGATACACACGTTAATGGGCAATGCGTCACC
ATTCTGACGTTTGCATCAGGCCAAAAAGTGGTATACAAACCTAGATCATTGTCG
ATAGATAAACAGTTCGGAGAATTCATCGAGTGGGTAAACTCGAAAGGTTTTCAG
CCTTCCTTGCGTATCCCTATTGCGATTGATCGTCAAACCTATGGTTGGTATGAA
TTCATCCCTCATCAAGAGGCCACCAGCGAAGATGAAATAGAACGCTACTATTCT
CGCATCGGTGGTTATCTGGCGATCGCCTACTTGTTCGGGGCAACCGACCTGCAC
CTGGATAACCTGATCGCCTGCGGCGAACATCCGATGCTTATTGATTTGGAAACA
CTCTTTACCAACGATCTCGACTGCTATGACAGTGCGTTTCCGTTCCCGGCGCTG
GCCCGCGAATTAACCCAATCCGTTTTTGGCACCCTTATGCTTCCCATCACCATC
GCGTCGGGGAAACTGCTGGATATAGACCTGTCAGCAGTAGGAGGCGGTAAAGGT
GTGCAGTCCGAAAAGATCAAAACCTGGGTCATCGTGAATCAGAAAACTGATGAG
ATGAAGCTGGTCGAGCAGCCGTATGTTACCGAGAGTTCCCAGAATAAACCAACA
GTTAATGGGAAAGAGGCGAACATTGGCAATTATATTCCTCATGTCACAGATGGC
TTTCGTAAAATGTACCGCCTGTTTCTGAATGAAATTGATGAGTTAATGGATCAT
AACGGGCCAATCTTTGCGTTTGAGAGTTGTCAGATTCGTCATGTTTTTCGAGCT
ACCCACGTGTATGCGAAATTTTTGGAGGCAAGTACCCACCCAGATTACTTGCAA
GAACCTACCAGACGTAATAAACTGTTCGAGTCCTTTTGGAACATCACGTCGCTG
ATGGCACCGTTCAAGAAAATTGTACCGCACGAAATCGCGGAGTTGGAGAACCAT
GATATTCCGTACTTCGTCCTGACTTGTGGCGGCACCATTGTTAAAGATGGATAC
GGCCGGGATATCGCAGACCTGTTTCAAAGTAGCTGCATCGAACGTGTAACTCAT
CGTCTGCAGCAGCTGGGAAGCGAGGATGAGGCGCGTCAAATTCGCTACATTAAA
AGCAGCCTGGCGACGTTGACCAACGGTGATTGGACCCCATCCCATGAGAAAACC
CCGATGTCTCCGGCCTCGGCCGACCGTGAAGATGGTTACTTCCTGCGCGAGGCT
CAGGCCATCGGCGACGACATTTTGGCGCAGCTGATTTGGGAGGATGACCGTCAC
GCCGCTTACCTTATTGGCGTAAGCGTGGGCATGAACGAAGCCGTCACTGTGTCA
CCCCTGACGCCTGGCATCTACGACGGCACACTTGGCATAGTGCTGTTCTTCGAT
CAGCTGGCCCAGCAGACCGGCGAAACCCATTATCGCCACGCCGCCGACGCTTTA
CTGGAAGGAATGTTCAAACAGCTGAAACCTGAACTGATGCCGTCTAGCGCTTAC
TTCGGACTGGGTAGCCTGTTCTATGGCCTGATGGTGTTGGGCCTCCAGCGTTCC
GACTCGCATATCATTCAGAAAGCGTATGAGTATCTGAAACATTTGGAAGAGTGT
GTGCAGCATGAGGAAACGCCAGATTTTGTCTCGGGTTTGTCTGGTGTACTGTAT
ATGCTCACGAAAATTTATCAGCTCACGAATGAACCGAGAGTTTTCGAAGTGGCC
AAAACCACAGCTTCGCGTCTGTCTGTGCTGCTTGACAGCAAGCAGCCCGACACT
GTGCTCACCGGGTTATCCCATGGCGCCGCAGGATTCGCCCTTGCATTACTGACC
TACGGAACCGCTGCAAATGATGAACAGTTGCTGAAACAGGGCCACTCCTATCTG
GTGTACGAACGTAATCGGTTTAACAAACAGGAAAACAACTGGGTTGATTTACGT
AAAGGCAACGCGTATCAAACATTTTGGTGCCATGGCGCCCCGGGTATTGGCATC
TCACGCCTCCTGTTAGCGCAATTTTACGATGACGAACTGCTGCATGAAGAGTTA
AACGCAGCACTGAACAAGACTATTTCGGACGGCTTCGGCCACAATCACTCACTG
TGTCATGGCGATTTCGGCAACCTCGATCTGTTATTGCTTTATGCCCAATATACG
AATAACCCAGAACCAAAGGAACTCGCTCGCAAACTGGCCATAAGCAGTATCGAT
CAAGCGCACACGTATGGCTGGAAACTCGGGCTCAATCATAGCGATCAACTGCAG
GGTATGATGTTAGGGGTGACTGGTATCGGCTATCAGCTCCTTCGTCATATAAAT
CCGACAGTCCCCAGCATTTTGGCACTGGAACTGCCCAGCTCCACGTTAACTGAA
AAAGAGCTGAGAATCCATGATCGTTGATAA
TGAAGGCCTCCCTAACGGGGGGCCTTTTTTTGTTTCTGGTCTCCCAAGCGCTTA
GGTCAACGCATGTGCTTTGCGTTCTGATGAGACAGTGATGTCGAAACCGCCTCT
GGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGAT
GTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAAC
GGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGG
CCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCG
GATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTA
CAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAA
GTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGAT
TTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCA
CACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTC
AAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAA
CAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTG
TCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTC
CTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTAC
AAATAA
TGAAGAGCGCAGAGGTGGTTGTGTTGCGAAAAAAAAAAAAAACACCCT
AACGGGTGTTTTTTTTTTTTTGGTGTCCCCACGTGTGGCGCTGGAGACCGTCCA
GGCAGAACGTGCAATGGAAACCCAGGGTAAACTGATTGCAGCAGCACTGGGTGT
TCTGCGTGAAAAAGGTTATGCAGGTTTTCGTATTGCAGATGTTCCGGGTGCAGC
CGGTGTTAGCCGTGGTGCACAGAGCCATCATTTTCCGACCAAACTGGAACTGCT
GCTGGCAACCTTTGAATGGCTGTATGAGCAGATTACCGAACGTAGCCGTGCACG
TCTGGCAAAACTGAAACCGGAAGATGATGTTATTCAGCAGATGCTGGATGATGC
AGCAGATTTTTTTCTGGATGATGATTTTAGCATCGGCCTGGATCTGATTGTTGC
AGCAGATCGTGATCCGGCACTGCGTGAAGGTATTCTGCGTACCGTTGAACGTAA
TCGTTTTGTTGTTGAAGATATGTGGCTGGGTGTGCTGGTGAGCCGTGGTCTGAG
CCGTGATGATGCCGAAGATATTCTGTGGCTGATTTTTAACAGCGTTCGTGGTCT
GACAGTTCGTAGCCTGTGGCAGAAAGATAAAGAACGTTTTGAACGTGTGCGTAA
TAGCACCCTGGAAATTGCACGTGAACGTTATGCAAAATTCAAACGTTGATAAGG
TTGCAGAATCCCTGCTTCGTCCATTTGACAGGCACATTATGCATCGATGATAAG
CTGTCAAACATGAGCAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCG
GCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAG
ATCGGGCTCGCCACTTCGGGCTCATGAGCAAATATTTTATCTGAGGTGCTTCCT
AATTAAGCCCCACTTCCACGTTGAGATAATTGAGCCGAAGCAAGTGTATCTCCT
GGGCGAACAGGGCAACCACGCTCTCACCGGGCAGCTCTACTGCCAAATTCTGCC
TTTCTTAAACGGCGAATACACCCGAGAACAAATTGTGGAAAAGCTCGATGGGCA
GGTCCCGGAGGAATATATCGACTTCGTACTCAGTCGTCTGGTGGAGAAGGGCTA
TCTAACTGAGGTGGCTCCAGAACTATCCCTGGAAGTGGCAGCATTTTGGAGCGA
ATTGGGAATTGCCCCTTCTGTAGTGGCAGAAGGGCTAAAGCAGCCAGTGACAGT
GACAACGGCGGGCAAGGGCATTAGGGAAGGGATAGTGGCTAACCTGGCAGCAGC
GCTGGAGGAAGCTGGCATTCAGGTGTCAGACCCAAGGGACCCAAAGGCCCCAAA
GGCAGGGGATTCTACTGCCCAGCTTCAGGTGGTGCTGACCGATGACTATTTACA
GCCGGAACTTGCAGCGATCAACAAGGAAGCCTTAGAGCGCCAACAACCCTGGTT
GCTGGTTAAGCCTGTGGGCAGTATCCTCTGGTTGGGACCGTTGTTCGTTCCTGG
GGAAACCGGATGTTGGCACTGTCTTGCTCAACGATTGCAAGGCAACCGGGAAGT
TGAAGCATCGGTATTGCAACAAAAGCGAGCGCTGCAGGAGCGCAACGGTCAAAA
TAAAAATGGTGCAGTGAGTTGCTTGCCCACAGCACGGGCAACCCTACCTTCTAC
TCTACAAACAGGTTTACAGTGGGCTGCCACTGAGATTGCTAAGTGGATGGTCAA
GCGGCACCTCAATGCCATAGCACCGGGAACGGCTCGTTTTCCCACTCTAGCTGG
CAAGATATTTACATTCAACCAGACGACTCTGGAGTTGAAAGCTCATCCTCTGAG
CCGACGACCGCAATGTCCCACCTGTGGCGATCGGGAAACTCTCCAACGGCGCGG
GTTTGAACCACTGAAGCTAGAGTCGCGCCCCAAACACTTCACCTCCGATGGCGG
TCATCGCGCCATGACCCCAGAACAAACGGTGCAGAAGTACCAACACCTCATCGG
GCCCATAACGGGGGTAGTGACGGAACTGGTGCGAATTTCTGACCCTGCCAATCC
CTTGGTGCATACCTACCGGGCTGGGCATAGCTTTGGCAGTGCTACGTCTCTGCG
GGGGCTGCGCAATGTCCTACGCCACAAGAGTTCTGGTAAAGGCAAGACCGATAG
CCAATCTCGGGCCAGCGGACTTTGCGAGGCGATCGAGCGCTATTCGGGCATTTT
TCAGGGAGACGAACCCCGCAAGCGGGCAACTTTGGCTGAGTTGGGAGATTTGGC
GATTCATCCAGAACAGTGTTTGCACTTTAGCGACAGGCAGTATGACAACCGGGA
AAGCTCGAACGAGCGAGCAACAGTGACTCACGACTGGATTCCCCAACGGTTCGA
TGCAAGTAAGGCTCACGACTGGACTCCCGTGTGGTCCCTAACGGAGCAAACCCA
TAAGTATCTGCCTACAGCCCTGTGCTATTACCGATACCCCTTCCCCCCAGAACA
CCGTTTCTGCCGTAGTGACTCCAACGGAAACGCGGCGGGAAATACCCTGGAAGA
GGCGATTTTGCAAGGATTTATGGAACTGGTGGAACGGGATAGCGTGTGCCTGTG
GTGGTACAATCGCGTTAGCCGTCCGGCTGTGGATTTGAGTAGCTTTGACGAGCC
TTATTTTTTGCAGTTGCAGCAGTTCTATCAAACTCAAAATCGCGATCTGTGGGT
ACTGGATTTAACAGCAGATTTGGGCATTCCGGCTTTTGTAGGGGTATCGAATCG
GAAAGCCGGCAGCTCGGAAAGAATAATTCTCGGCTTTGGAGCGCACCTGGACCC
GACAGTTGCCATCCTTCGCGCTCTTACGGAGGTCAACCAAATAGGCTTGGAATT
GGATAAAGTTTCTGATGAGAGCCTCAAGAACGATGCCACGGATTGGTTAGTGAA
TGCTACATTGGCAGCTAGTCCCTATCTCGTTGCCGATGCTAGCCAACCCCTCAA
GACTGCGAAGGATTATCCCCGGCGTTGGAGTGACGATATTTACACCGATGTGAT
GACTTGTGTAGAAATAGCCAAGCAAGCAGGTCTAGAGACTTTGGTACTGGATCA
GACCAGACCCGACATAGGTTTAAATGTGGTTAAAGTCATTGTGCCAGGAATGCG
TTTTTGGTCGCGATTTGGCTCCGGTCGGCTCTATGACGTGCCAGTGAAGTTGGG
ATGGCGAGAGCAACCACTTGCTGAGGCACAAATGAACCCTACACCGATGCCATT
TTAATAA
ATTTATTAATGAAAGTGTAAGAGTTCACCAGCTTCCTGAGGGCGGCGTGTTAGA
AATCGACTACTTGCGCGATAATGTCTCCATTTCTGACTTTGAGTATTTGGATCT
CAACAAAACGGCTTACGAGCTCTGCATGCGCATGGATGGCCAAAAAACAGCTGA
GCAGATTTTAGCTGAGCAATGTGCAGTGTATGATGAATCACCGGAAGATCATAA
AGATTGGTATTACGACATGCTCAACATGCTCCAGAACAAGCAGGTTATTCAGCT
TGGAAACCGGGCCAGCCGCCATACAATCACCACGAGCGGAAGCAATGAATTTCC
GATGCCCCTGCACGCCACCTTTGAACTGACGCACCGCTGTAATTTGAAATGCGC
CCACTGTTATTTGGAAAGCTCACCTGAAGCGCTCGGCACCGTGTCGATTGAGCA
ATTCAAAAAAACGGCTGATATGCTGTTTGATAACGGTGTATTGACATGCGAAAT
CACAGGTGGAGAAATTTTTGTCCATCCAAACGCCAATGAGATTCTTGACTATGT
GTGTAAAAAGTTCAAAAAAGTCGCTGTCTTAACAAACGGAACACTCATGCGAAA
AGAGAGCCTGGAGCTTTTGAAAACTTACAAGCAAAAAATCATCGTCGGCATTTC
TCTAGATAGTGTCAATTCCGAGGTCCATGACTCCTTTAGAGGGAGAAAAGGCTC
TTTTGCCCAAACTTGTAAAACGATAAAATTGTTGAGTGACCACGGTATATTTGT
CAGAGTCGCTATGTCTGTATTCGAAAAAAACATGTGGGAAATCCACGATATGGC
CCAAAAGGTTCGGGATCTCGGGGCGAAGGCGTTTTCTTACAATTGGGTTGACGA
TTTCGGAAGAGGCAGGGATATTGTCCATCCAACGAAAGACGCCGAGCAGCACCG
CAAGTTTATGGAATACGAGCAACATGTGATTGATGAGTTTAAAGATCTGATTCC
GATTATTCCCTATGAGAGAAAACGCGCGGCAAATTGCGGCGCTGGCTGGAAGTC
CATTGTGATCAGTCCGTTCGGCGAAGTACGTCCTTGCGCCCTCTTTCCAAAGGA
ATTTTCATTGGGAAATATTTTTCATGATTCCTATGAAAGCATCTTTAACTCCCC
TCTCGTCCATAAACTGTGGCAAGCGCAAGCGCCGCGGTTCAGCGAACATTGCAT
GAAAGACAAATGCCCGTTCAGCGGCTATTGCGGAGGCTGTTACTTAAAAGGGCT
GAACTCTAACAAATATCACCGGAAAAACATTTGCTCTTGGGCGAAAAATGAACA
ATTAGAAGATGTGGTCCAGCTTATTTAGTAA
CTTTTAGCCACGATAATGAAAGTATTCCTCTGGTAATCAAAGCCATAGAAGCCA
TGGGTAAAAAAGCCTTCCGTTTTGATACTGATCGCTTCCCTACAGAGGTGAAAG
TTGATCTTTACTCAGGCGGTCAAAAAGGCGGAATTATTACCGATGGAGAACAAA
AATTAGAGCTAAAAGAAGTTTCTTCTGTCTGGTATCGACGCATGAGATACGGAC
TAAAATTACCCGATGGGATGGATAGTCAATTTCGCGAAGCTTCTCTTAAGGAAT
GTCGGTTAAGTATTCGAGGAATGATTGCTAGTTTATCTGGCTTTCATCTTGATC
CAATTGCTAAGGTAGATCATGCTAATCATAAACAATTGCAGTTACAAGTGGCGC
AACAATTAGGTTTATTAATTCCGGGGACTTTAACTTCTAATAATCCTGAAGCTG
TCAAGCAATTTGCTCGGGAGTTTGAAGCGACGGGAATTGTGACTAAAATGCTTT
CTCAATTTGCTATTTATGGAGACAAGCAAGAGGAAATGGTTGTTTTTACCAGTC
CTGTTACAAAGGAAGATCTAGATAATTTGGAAGGTTTGCAATTTTGTCCAATGA
CTTTTCAGGAAAACATTCCTAAAGCTTTGGAATTACGCATCACTATCGTCGGTG
AACAAATATTTACGGCGGCGATTAATTCCCAACAATTAGACGGTGCTATCTACG
ATTGGCGAAAAGAGGGACGCGCGCTCCATCAACAATGGCAACCCTACGATTTAC
CGAAAACTATTGAAAAACAACTACTAGAATTAGTGAAATATTTCGGTCTTAATT
ATGGTGCAATTGATATGATTGTCACACCAGATGAACGTTATATCTTTTTAGAAA
TTAATCCCGTTGGCGAGTTTTTCTGGCTAGAACTTTATCCTCCTTATTTTCCTA
TCTCCCAGGCGATCGCTGAAATCCTAGTTAACTCATAATAA
GAAAACATCGTGGCTGGCCGCCATCGCTCCGGATGAACCCCACAAATTCGACCG
CCGCTTAGAATGGGACGAGCTTTCAGAGGAGAACTTCTTCGCAGCACTGAACTC
AGAACCTGCATCGTTGGAAGAGGATGATCCATGTTTTGAAGAAGCACTGCAAGA
CGCCCTGGAGGCCTTGAAGGCAGCATGGGATTTACCCCTTCTTCCCGTCGATAA
TAATCTTAATCGTCCCTTCGTAGATGTCTGGTGGCCCATTCGCTGTCACTCTGC
GGAGAGCTTGCGTCAAAGCTTCGTCAGTGATAGTGCTGGACTTGCGGACGAGAT
TTTTGATCAGCTGGCCGATTCGTTACTGGACCGTCTGTGCGCCCTGGGAGATCA
GGTGTTGTGGGAGGCGTTTAACAAGGAGCGTACACCAGGAACGATGTTGTTAGC
CCACTTAGGAGCCGCAGGCGACGGCTCCGGACCCCCTGTACGTGAGCATTACGA
ACGTTTTATTCAGTCTCACCGCCGTAATGGATTAGCGCCTTTGCTTAAGGAATT
CCCTGTACTGGGCCGCCTTATTGGAACAGTTTTGTCCCTTTGGTTCCAAGGGAG
CGTGGAAATGCTGCAACGTATCTGCGCTGACCGCACCGTTCTGCAACAGTGTTT
CGCTATCCCTTGCGGGCATCACCTGAAAACTGTAAAGCAGGGACTTTCTGATCC
ACACCGCGGCGGTCGCGCTGTGGCAGTTTTGGAATTTGCGGACCCAAATTCCAC
CGCTAATTCAAGTATGCACGTAGTGTATAAACCGAAGGATATGGCTGTGGATGC
AGCTTACCAGGCCACCTTAGCAGATCTTAATACTCATAGCGACCTTTCCCCGTT
GCGCACGCTTGCCATTCATAACGGCAACGGATATGGTTACATGGAACATGTGGT
TCACCATCTTTGCGCTAACGACAAAGAGCTGACAAATTTCTATTTCAACGCTGG
GCGTTTAACCGCGCTTCTGCATCTTCTTGGATGTACTGACTGTCACCATGAAAA
TTTGATTGCATGTGGTGATCAATTACTGTTGATCGATACAGAAACATTATTGGA
GGCGGATTTACCCGATCACATTTCGGATGCTTCGAGCACCACGGCGCAACCAAA
GCCTAGTAGCCTTCAAAAGCAATTTCAGCGTTCTGTTTTGCGTAGCGGGTTACT
TCCTCAATGGATGTTCCTGGGGGAGTCGAAGTTGGCCATCGACATCTCGGCTCT
GGGAATGTCCCCACCCAATAAGCCTGAGCGTATTGCACTTGGCTGGTTAGGATT
CAATTCTGACGGGATGATGCCTGGGCGTGTATCCCAACCAGTTGAGATTCCTAC
ATCCTTGCCCGTTGGGATTGGTGAGGTTAATCCCTTTGATCGTTTTTTAGAGGA
TTTTTGTGATGGCTTTTCCATGCAATCAGAGGCCCTTATTAAGCTTCGCAACCG
TTGGCTGGACGTTAATGGGGTTCTTGCTCATTTCGCGGGTCTGCCCCGCCGTAT
CGTTCTTCGCGCGACTCGCGTATACTTCACTATCCAGCGTCAGCAGTTAGAGCC
TACGGCACTGCGCTCTCCACTTGCACAGGCCTTGAAACTTGAGCAGCTTACTCG
TTCTTTCTTGTTGGCAGAGTCAAAGCCTCTTCACTGGCCCATTTTCGCAGCTGA
AGTAAAGCAGATGCAGCACCTTGACATTCCTTTCTTCACACACTTAATCGACGC
TGACGCTCTGCAGCTGGGCGGCCTGGAACAAGAATTACCAGGCTTCATCCAGAC
TAGTGGCTTGGCAGCTGCTTACGAGCGTTTGCGTAATTTAGATACGGACGAGAT
TGCTTTCCAACTTCGTCTGATCCGCGGTGCAGTAGAGGCTCGCGAGTTGCATAC
TACGCCGGAGTCGAGCCCGACGTTGCCGCCGCCTGCCACCCCCGAGGCTCTTAT
GTCCTCTTCAGCCGAGACTAGTTTAGAAGCTGCTAAGCGCATCGCTCACCGCTT
ACTGGAGTTGGCAATTCGTGATTCTCAAGGGCAAGTAGAATGGCTGGGCATGGA
TCTGGGGGCAGATGGAGAGAGCTTCTCCTTTGGCCCAGTTGGCTTGAGCCTTTA
TGGGGGCTCAATCGGTATCGCTCACCTTCTGCAACGTTTGCAGGCGCAGCAAGT
TTCCTTGATGGACGCAGACGCTATCCAAACGGCAATTTTACAGCCCCTTGTGGG
ACTGGTTGATCAACCTAGCGACGACGGACGTCGCCGTTGGTGGCGTGATCAGCC
GCTGGGCTTAAGTGGATGTGGCGGTACCTTGCTTGCACTTACACTTCAAGGTGA
ACAAGCGATGGCTAATTCCCTGCTGGCCGCTGCTTTGCCCCGTTTTATCGAGGC
TGATCAGCAACTTGACCTGATTGGTGGCTGCGCTGGACTGATCGGTTCGTTGGT
ACAATTAGGTACTGAAAGTGCCTTACAATTAGCTTTGCGTGCGGGCGACCATCT
TATTGCGCAACAGAATGAAGAGGGGGCGTGGTCTAGCTCGTCATCACAGCCCGG
TTTGTTGGGCTTTAGTCATGGTACTGCAGGTTACGCAGCAGCCTTAGCACACTT
ACATGCATTTTCCGCTGATGAGCGTTACCGCACCGCAGCCGCTGCCGCTTTAGC
ATACGAACGCGCACGTTTTAATAAAGATGCCGGCAACTGGCCAGACTACCGCTC
GATCGGACGTGACTCTGATTCAGATGAACCGTCCTTTATGGCTTCCTGGTGTCA
CGGCGCACCCGGCATTGCCCTGGGCCGCGCCTGTTTGTGGGGTACGGCGCTTTG
GGACGAAGAATGCACCAAGGAGATCGGAATTGGGTTACAGACCACAGCTGCTGT
TTCGTCTGTTAGTACTGACCACCTGTGTTGTGGTTCACTTGGCCTTATGGTATT
ATTAGAGATGCTGTCAGCAGGACCCTGGCCCATCGACAATCAATTACGTTCCCA
TTGCCAGGACGTAGCATTCCAGTACCGCCTGCAGGCTTTGCAGCGCTGTTCAGC
CGAGCCGATTAAGCTTCGTTGCTTCGGTACAAAAGAGGGCCTTTTAGTCCTGCC
TGGATTTTTCACTGGCTTATCAGGAATGGGTTTAGCACTGCTTGAGGATGATCC
ATCTCGCGCCGTGGTTTCTCAACTGATCAGTGCGGGCTTATGGCCGACAGAGTG
ATAA
CTCTGGCACGTCTGTTTGACGTGTTGGGTGACGATGCCGCTGCCGCACGTGAAT
GGGTAACGGAACCCCATCGTCTGATCGCTAGCAATGAGCGCCTGGGCACAGCTC
CGGAAGCCCCGGCGGATGACGATCCGGAGGCCATTCGGACGGTTGGAGTGATCG
GAGGGGGCACAGCCGGGTATTTAACGGCGTTGGCTCTGAAGGCTAAACGCCCTT
GGTTGGATGTGGCGCTCGTCGAAAGTGCGGATATCCCGATCATTGGGGTAGGAG
AGGCGACGGTGTCTTATATGGTGATGTTTCTGCACCATTATCTGGGCATTGATC
CGGCGGAGTTTTACCAACATGTGCGCCCTACTTGGAAACTGGGCATCCGTTTTG
AATGGGGGTCACGTCCGGAGGGCTTTGTTGCGCCATTCGATTGGGGGACCGGAT
CTGTTGGCCTGGTTGGGAGCCTGCGTGAAACGGGCAATGTCAACGAAGCTACGT
TACAGGCGATGCTCATGACGGAGGATCGCGTTCCGGTATATCGTGGCGAAGGTG
GGCATGTTAGTCTGATGAAATATCTGCCATTCGCATATCATATGGATAACGCTC
GCCTGGTTCGCTACCTGACGGAACTCGCCACTCGTCGTGGCGTGCATCATGTCG
ATGCGACTGTAGCTGAAGTTCGCCTGGATGGTCCTGACCACGTTGGGGACCTGA
TTACTACGGACGGTCGTCGCCTGCACTATGACTTTTACGTCGATTGTACTGGAT
TTCGTTCCCTGCTGCTGGAAAAAGCCCTGGGTATCCCGTTCGAATCTTATGCGT
CAAGCCTGTTTACCGACGCGGCAATTACCGGTACCCTTGCACATGGGGGTCATC
TTAAACCTTACACTACGGCAACTACCATGAATGCGGGCTGGTGTTGGACGATCC
CTACTCCTGAGTCCGATCACCTGGGGTACGTTTTCAGTAGTGCCGCGATCGATC
CAGACGATGCAGCAGCAGAAATGGCCCGCCGTTTCCCGGGCGTTACCCGCGAAG
CATTAGTTCGCTTTCGCTCCGGCCGTCACCGTGAAGCTTGGCGCGGCAATGTCA
TCGCGGTAGGAAACAGCTATGCTTTCGTGGAACCTCTGGAGAGTTCGGGACTCC
TGATGATTGCTACCGCAGTCCAGATCCTGGTGAGTTTGCTGCCGAGTAGTCGTC
GTGACCCGCTGCCTAGCAATGTGGCGAATCAGGCGTTAGCTCACCGGTGGGACG
CGATTCGTTGGTTTCTGAGTATTCATTACCGTTTCAACGGCCGCCTCGATACTC
CGTTCTGGAAGGAAGCCCGTGCCGAAACAGATATTAGCGGTATTGAACCGTTGC
TTCGTCTGTTCAGTGCCGGTGCCCCTCTGACCGGTCGCGATAGCTTTGCGCGCT
ATTTGGCCGACGGAGCAGCCCCGTTGTTCTATGGCCTGGAGGGTGTTGATACCT
TACTGCTGGGACAGGAAGTGCCTGCGCGTCTGTTACCACCGCGTGAATCTCCTG
AGCAGTGGCGTGCCCGTGCTGCAGCAGCCCGCTCATTAGCCTCGCGTGGCTTAC
GTCAGAGCGAAGCTCTGGATGCTTACGCTGCGGACCCCTGTCTCAATGCGGAAC
TGCTGTCTGATAGCGACTCATGGGCGGGTGAACGCGTCGCGGTACGTGCAGGTC
GACGACTGGCACCACGGTAGCGCATGCTGTAGAACCAGACGGTTTCCGCGCCGT
GATGGCCACACTGCCGGCCGCTGTGGCGATCGTTACGGCAGCTGCGGCAGATGG
GCGCCCGTGGGGTATGACCTGCAGTTCGGTTTGCTCAGTGACCTTGACCCCGCC
GACCCTTCTGGTCTGCCTTCGGACGGCGTCCCCGACTCTGGCCGCAGTCGTGTC
AGGTCGTGCATTTAGCGTGAACCTTCTGTGTGCGCGGGCCTATCCTGTGGCGGA
ATTGTTTGCATCTGCGGCAGCAGACCGGTTTGATCGCGTTCGTTGGCGTCGCCC
GCCGGGTACAGGCGGTCCACATCTTGCCGATGATGCACGTGCAGTGTTAGACTG
TCGCCTGAGCGAAAGCGCAGAAGTAGGCGACCATGTGGTCGTATTTGGCCAAGT
CCGGGCGATTCGTCGCCTGAGTGATGAACCACCACTGATGTATGGTTATCGTCG
TTACGCACCTTGGCCGGCAGATCGTGGTCCGGGTGCGGCAGGCGGCTAATAA
AGCGACGCAGGAGGTGACCCACGCCCGCCTGAACGCTTACTGTTGGGGGTGTCA
GGAAGTGTCGCTGCACTGAACTTACCGGCGTACATTTATGCCTTTCGGGCAGCC
GGTGTGGCACGTCTTGCGGTCGTGCTGACACCAGCGGCTGAAGGGTTCCTTCCA
GCGGGTGCGTTACGCCCGATTGTGGATGCCGTTCATACGGAACATGACCAAGGC
AAAGGTCACGTAGCGCTGTCACGCTGGGCGCAACACTTACTCGTGCTGCCGGCA
ACAGCGAATTTGCTTGGCTGTGCAGCGTCAGGACTTGCGCCGAACTTTTTAGCG
ACCGTTCTGCTCGCGGCAGATTGCCCAATCACATTCGTCCCGGCGATGAATCCG
GTCATGTGGCGTAAACCAGCCGTACGCCGGAACGTTGCAACCTTACGCGCAGAT
GGTCATCACGTGGTGGATCCTCTGCCGGGCGCTGTGTACGAAGCTGCCTCACGT
TCTATCGTGGAAGGTCTTGCTATGCCGCGCCCTGAAGCGTTAGTCCGTTTACTG
GGTGGCGGTGATGACGGTTCTCCAGCAGGACCGGCAGGTCCGGTTGGACGCGCA
GAGCATGTTGGGGCTGTTGAGGCTGTTGAAGCCGTGGAAGCAGTTGAGGCCGTT
GAGGCTGCGGAAGCACTTGCGTAATAA
TCCTACGCAGTGTGGGAAATCACCCTGAAATGCAATCTGGCATGCTCTCATTGT
GGCAGCCGCGCCGGCCAAGCCCGTACGAAAGAGCTGAGTACCGAAGAAGCGTTC
AACCTGGTCCGCCAGCTGGCCGACGTGGGCATTAAGGAAGTCACCCTGATCGGT
GGTGAAGCCTTTATGCGTTCGGATTGGCTGGAAATCGCGAAAGCCGTCACTGAA
GCCGGCATGATCTGTGGCATGACCACAGGGGGCTTCGGGGTCAGTCTGGAAACG
GCGCGTAAAATGAAAGAAGCGGGCATTAAAACGGTGAGCGTTAGCATTGACGGT
GGTATTCCTGAAACCCACGACCGCCAGCGCGGTAAAAAGGGTGCGTGGCATAGT
GCATTCCGGACTATGAGCCATCTGAAAGAAGTCGGGATCTACTTCGGTTGCAAC
ACTCAAATCAATCGTTTATCGGCGTCAGAATTCCCGATTATCTATGAACGTATT
CGCGATGCTGGGGCACGTGCGTGGCAAATTCAGCTGACGGTTCCGATGGGCAAC
GCCGCGGATAACGCAGATATGCTGCTGCAACCGTATGAATTGCTCGACATCTAT
CCGATGTTAGCCCGCGTTGCCAAACGTGCGAAACAGGAAGGCGTGCGTATTCAG
GCAGGTAACAACATCGGGTACTATGGACCGTATGAGCGTCTGCTGCGTGGCAGC
GACGAATGGACGTTTTGGCAAGGATGTGGTGCGGGCCTTAACACCCTCGGCATC
GAAGCCGACGGCAAAATCAAAGGCTGTCCATCCCTGCCGACCGCCGCGTACACC
GGCGGTAACATTCGCGATCGCCCGCTGCGGGAAATCGTCGAACAGACCGAAGAA
CTGAAATTTAACTTAAAAGCTGGTACAGAACAAGGTACGGACCATATGTGGGGC
TTTTGTAAAACCTGCGAATTCGCGGAACTCTGTCGCGGCGGATGCAGCTGGACT
GCGCATGTGTTCTTTGACCGGCGCGGCAATAATCCGTACTGCCACCATCGGGCT
CTGAAACAAGCCCAAAAAGACATTCGCGAACGCTTTTATTTAAAAGTGAAAGCA
AAGGGCAACCCGTTCGACAATGGTGAATTTGTTATCATTGAAGAACCTTTTAAC
GCTCCGTTACCCGAGAATGACCTGCTGCACTTTAACAGTGATCACATTCAATGG
CGCGGCACAGAAATCTGACGACAGCAGCAGCGTATTACCGCGCCAGGGGTGGCA
AGACAAACAAGCCTTTATTAAGGCACTCATTAAAGCCAAACAGTCTCTCGAAAT
TGCCGAAATTAGCAACTTTTTAACC
ATCCCCTGTCGCGTCCAGAACCGCTGGGCGTGCACCCAGATTATCGTCGCCTGC
GTGAGACTTGCCCGGTTGCACGTGTGGGTAGCCCGTATGGCCCAGCGTGGCTTG
TCACCCGTTACGCCGATGTGGCCGCAGTTCTGACCGATGCCCGTTTTAGTCGTG
CAGCCGCTCCGGAAGATGATGGTGGCATCCTGCTGAACACCGATCCGCCGGAAC
ATGATCGTCTGCGTAAACTGATTGTAGCACACACAGGCACCGCTCGCGTGGAAC
GGCTGCGTCCGCGTGCTGAAGAGATCGCTGTTGCGTTAGCGCGCCGTATCCCGG
GCGAAGGCGAATTCATTAGTGCATTTGCCGAGCCCTTCAGCCATCGCGTTTTGT
CTTTATTTGTTGGCCATCTTGTTGGGTTACCAGCGCAGGACCTGGGCCCCTTAG
CGACCGTAGTGACTCTGGCACCCGTTCCCGACCGCGAACGTGGCGCGGCATTTG
CAGAGCTGTGTCGTCGGCTGGGTCGTCAGGTGGATCGCGAAACGCTTGCAGTAG
TTTTAAACGTGGTCTTTGGCGGACATGCGGCTGTAGTGGCCGCGCTGGGTTATT
GCCTGTTAGCTGCATTAGATGCGCCACTGCCACGTCTGGCCGGTGACCCAGAGG
GCATTGCCGAACTGGTGGAAGAAACCCTTCGTTTGGCTCCACCGGGAGATCGTA
CACTGTTGCGTCGTACTACAGAACCTGTGGAACTTGGCGGTCGCACATTACCAG
CGGGTGCGCTTGTAATCCCGTCCATTGCAGCCGCAAACCGTGATCCGGATCGCC
CTGTGGGCCGTCGTATGCCACGTCATCTTGCATTTGGACGTGGAGCGCATGCCT
GTTTAGGCATGGCGCTGGCGCGCATGGAACTCCAGGCAGCACTGAAAGCGTTAG
CGGAACACGCGCCAGACGTACGGTTGCCGGCTGGTACAGGCGCGCTGGTCCGCA
CACACGAAGAACTCTCGGTGAGCCCGCTCGCAGGAATCCCAATTCAACGCTAAT
AA
TCGATGAAGCTGCGGTGGCGGCGGACTTACGCGAATTGGCCGCAGCTCTGGATC
GCAGTGGTTATGGTGAAATCCTCACCTGTTTTCTGCCTCAGAAGGCACAGGCGC
ATATCTGGGCTCAGACCGCTGCAAAAATTGATGGGCCGTTGCGTACCCTGATGG
AATTATTCCTTCTGGGTCGGGCGGTTCCCCAGGATGATCTCCCGCCTCGCATCG
CGGCCGTGATTCCCGGTTTAGTTAGCGCAGGTCTGGTTAAGACTGGACAGGGCG
CGGTTTGGCTGCCGAACTTGATTCTGCTGCGTCCTATGGGCCAGTGGTTATGGT
GTCAGCGGCCTCACCCCTCACCGACCATGTACTTTGGTGACGATAGCCTGGCGC
TGGTTCACCGGATGGTAACATATCGTGGCGGCCGTGCCCTGGATTTATGTGCAG
GTCCGGGTGTTCAGGCCCTTACCGCAGCCCTCCGCTCAGAGCACGTTACCGCGG
TTGAGATCAATCCGGTCGCGGCAGCCCTTTGCCGCACCAACATTGCCATGAACG
GTCTGTCCGACCGCATGGAGGTTCGCCTGGGCTCACTGTACGACGTCGTGCGCG
GTGAGGTTTTTGATGATATTGTATCAAACCCGCCGCTGCTGCCTGTTCCGGAGG
ATGTGCAATTCGCCTTTGTGGGAGATGGCGGACGCGATGGTTTCGATATTTCTT
GGACGATTCTGGATGGCCTGCCTGAACATCTGTCCGACCGTGGTGCGTGTCGCA
TCGTTGGTTGTGTTCTGTCCGATGGCTATGTGCCTGTTGTGATGGAAGGCTTGG
GAGAATGGGCCGCTAAACACGATTTCGACGTGCTTCTTACAGTGACCGCACATG
TCGAGGCGCATAAAGATAGTAGTTTTCTGCGTTCAATGAGCCTGATGAGTTCGG
CGATCTCAGGCCGCCCAGCGGAGGAGCTGCAAGAACGGTACGCAGCTGATTATG
CCGAACTGGGCGGTTCCCACGTTGCGTTCTATGAACTGTGTGCCCGCCGTGGTG
GGGGTTCTGCACGTCTGGCCGACGTGAGCGCTACAAAACGCAGTGCGGAAGTGT
GGTTTGTTTAATAA
TTAAAGAATCCCACCACATCATTTTAGCTGACGATGGTGACATTTGCATTGGGG
AAATTCCGGGGGTGTCTCAGGTAATCAATGACCCGCCGTCGTGGGTTCGTCCTG
CCCTGGCAAAGATGGATGGCAAGCGTACTGTCCCCCGTATTTTCAAAGAACTGG
TCAGTGAAGGCGTACAGATCGAATCCGAACATCTGGAAGGCCTGGTAGCCGGGC
TTGCCGAACGCAAACTTCTCCAGGATAACAGTTTCTTTTCCAAGGTGTTAAGCG
GTGAAGAAGTGGAGCGCTATAACCGCCAGATTCTGCAGTTCAGCCTTATCGATG
CGGATAACCAGCACCCTTTCGTTTACCAAGAGCGGCTGAAACAGTCTAAAGTCG
CTATCTTCGGTATGGGTGGCTGGGGCACGTGGTGTGCATTGCAGCTGGCCATGT
CAGGCATTGGTACACTGCGGCTGATCGACGGCGATGATGTGGAACTGTCGAACA
TTAACCGCCAAGTTCTGTATCGCACGGATGATGTAGGTAAAAACAAAGTTGATG
CCGCCAAAGACACTATCCTGGCATACAACGAAAACGTGCATGTTGAAACCTTCT
TTGAATTCGCCAGCCCGGACCGTGCCCGGCTTGAAGAACTTGTGGGTGATTCTA
CCTTTATTATCCTGGCTTGGGCCGCGTTGGGTTACTACCGTAAAGATACGGCAG
AGGAAATTATCCATTCGATTGCGAAAGATAAAGCGATCCCTGTAATTGAACTCG
GCGGTGATCCTTTGGAAATCTCTGTCGGTCCTATTTACCTGAATGATGGCGTAC
ACAGCGGCTTCGACGAGGTGAAAAATTCCGTTAAAGATAAATACTACGACAGCA
ACAGCGATATCCGCAAATTTCAAGAGGCGCGGTTGAAACACAGCTTCATCGATG
GCGATCGTAAAGTGAACGCGTGGCAATCAGCGCCCAGCCTGAGTATTATGGCTG
GTATCGTAACGGATCAGGTTGTGAAAACCATTACCGGGTACGACAAGCCACATC
TCGTTGGCAAGAAATTTATCTTGAGTCTGCAAGATTTCCGCAGCCGCGAGGAGG
AGATCTTTAAATAATAA
TTCTGCGCGATGCGTTAGATCCGGATCGCTTCGGCCGCGAGATGAAGGCAGTAA
CAGAAATTCCCGAGATCGTTAAACTCGGCCATCGTCATGGTTATGGATTTACTG
CCGAAGAATTTCTGACCAAAGCTATGAGTTTTGGTGCTCCGCCGGCAGGAGCAG
CAGCACCTGGCGAATCAGCCAGCGTTCCTGGCCAGAACGGTTCCTCCCCCGGAC
ACGCTGCGCGTGCAGCTATGGCTGGTCCAGAAGCAGGGGCCACCAGCTTTGCCC
ACTATGAATACCGTCTGGATGAGCTGCCGGAATTCGCCCCCGTTGTGGCCGAGC
TTCCGAAACTGAAAGTCATGCCGCCTTCCGTGGGACCTGATCGGTTTGCAGCAC
GCTACCGTGATGAAGATATGCGCACAATTTCAATGAGTCCGGCGGATCCGGCTT
ACCAGGCTTGGCACCAGGAACTGGCGGGTCGTGGTTGGCGCGATGCAGAAGATA
CGGCTGCTGCTCCAGATGCCCCACGGCGCGATTTTCATCTGCTGAACCTCGATG
AGCATGTAGATTACCCAGGTTATGAAGAATATTTTGCGGCCAAGACCCGTGTCG
TCGCGGCACTCGAAAACCTGTTTGGTGGTGACGTGCGTTGCTCAGGCTCTATGT
GGTATCCGCCGTCGAGCTATCGCTTATGGCATACAAATGCCGATCAACCGGGGT
GGCGTATGTACCTGGTAGATGTAGATCGCCCATTCGCGGACCCCGACCGTACCT
CCTTCTTTCGCTACCTGCATCCACGTACCCGTGAAATCGTCACGCTGCGCGAAA
GCCCTCGTATTGTCCGTTTCTTTAAAGTCGAACAGGATCCCGAGAAGCTGTTCT
GGCACTGTATCGCGAACCCCACCGATCGCCATCGCTGGTCGTTTGGTTACGTTG
TTCCGGAAAACTGGATGGACGCCCTCCGTCACCATGGCTAATAA
CCTGGAGGTTGTTGATGTTCGTCGCGGCGAGTCGTTCAAGGCATGGTCGCATGG
GTACCCATATCGCACTGTTCGCTGGCACTTCCATCCTGAGTTTGAAGTACATCT
GATCGTGGAAACCACCGGCCAGATGTTTGTGGGTGATTATGTCGGAGGCTTTGG
TCCGGGTAATCTGGTCCTGATGGGTCCCAATCTGCCTCATAATTGGGTGTCTGA
CGTTCCTGAGGGTAAAACCGTTGCAGAGCGTAACCTTGTTGTTCAATTTGGGCA
AGCGTTCGTTTCCCGTTGCGAGGATTCCTTAACGGAGTGGCGTCACGTGGAAAC
GTTACTGGCGGATGCGCGGCGTGGCGTGCAATTTGGGCCGCGCACCTCTGAGGC
CATTAAACCTCTGTTCGCGGAACTGATTCACGCGCGCGGCCTGCGTCGCATTGT
GCTGTTTCTGTCTATGCTGCAAATCCTCGTCGATGCAACGGATCGCGAACTGCT
GGCATCTCCAGCTTATCAGGCGGATCCTTCGACATTTGCAAGCACGCGCATTAA
TCATGCGCTGGCCTACATTGGAAAGAATCTGGCGAACGAGCTTCGTGAAACAGA
TTTAGCACGGCTGGCCGGACAGTCTGTTTCCGCCTTCTCTCATTATTTTCGTCG
TCATACCGGCCTGCCTTTCGTGCAGTACGTTAATCGCATGCGTATCAACCTGGC
CTGTCAGCTTCTGATGGACGGGGACGCATCGGTGACAGATATTTGTTTCCGTAG
CGGTTTTAACAACCTGTCCAATTTTAACCGTCAGTTTCTGGCAGTGAAAGGTAT
GTCACCCAGTCGGTTCCGTCGCTACCAGGCTCTCAACGACGCGTCACGTGATGC
GAGTGAAGCGGCTGCAAAACGCGGCGCAGGTATTGCAGGTGCACCGGCAATCGT
TCCAGCGGCTCAAGCACGTGGCGAGGCACGCCCAATTCCTGAAGTGCTGCTTAG
TGACGGCGAGCTCCACACCGGCATCCGGTAATCCAGCTGCCCGTGCATTGCGCG
CCGCTGCCTTTGCACTGGCCTTAGGCGGAGCATGCGTTGCGCATGCCGCACCTC
TGCGGATTGGCATGACATTCCAAGAATTGAATAACCCGTATTTTGTGACCATGC
AGAAAGCACTGAACGAAGCCGCGGCGAGCATTGGCGCGCAAGTGATTGTAACAG
ACGCACATCACGACGTGTCAAAACAGGTATCAGACGTTGAGGATATGCTGCAGA
AGAAAATTGATATTTTACTGGTGAATCCAACCGACTCCACGGGCATCCAGAGTG
CGATTGTTTCCGCAAAGAAGGCTGGCGCCGTGGTCGTGGCGGTCGATGCCAATG
CCAATGGCCCGGTGGATTCCTTCGTAGGGTCCAAGAATTTTGATGCCGGCGCTA
TGTCATGCGAGTACCTTGCGAAAGCGATCAACGGCGGCGGCGAAGTGGCCATTC
TGGATGGCATCCCGGTCGTCCCAATCCTGGAACGTGTCCGCGGCTGCCGCGCGG
CACTGGCCAAATTCCCGAATGTGAAAATTGTCGACGTTCAGAATGGAAAACAGG
AACGTGCGACAGCGTTAACGGTAACCGAGAATATGATCCAGGCGCACCCGAAAC
TGAAAGGTGTGTTTAGTGTAAACGACGGCGGGTCAATGGGCGCTTTGAGCGCCA
TTGAAGCGAGCGGCAAAGATATCCGCCTCACGTCCGTAGATGGTGCCCCAGAGG
CGGTGGCGGCGATTCAAAAGCCGAACTCCAAATTTATTGAAACAAGCGCTCAAT
TTCCGCGCGACCAGATCCGTTTAGCGATTGGTATTGGCCTGGCCAAGAAATGGG
GCGCGAACGTGCCAAAAGCGATTCCAGTCGACGTGAAACTGATTGACAAAGGGA
ACGCGAAAACCTTTAGTTGGTAATAA
ACAGACCGGTTTTGTTGTACTGCCAGACAACGATGCCACCGGCGACGTGACGGG
CCGCCTGTTACCTTGGGGTGATGTAGTTACAGTGTATCCGTCTGGCCGTCCATG
GATCATCGGCAACTGCTGGGATCGCCCAGTCCTCGTCCATGATGGCGTGATCGT
CTTGGGTCATACCAGCGTCACGCGTGATCAAATTGCCCGTCATGGGAACGATCC
GCATCGCTTACTGGACGAGGCCGACGGCGCATTTCATGCGGCGGTCCTGATCGG
ACACGAAGTTCATGTTCGCGGCTCCGCCTACGGTGTCTGTCGTCTGTATACATG
CGTTGTTGACGGTGTGACCTTAGTGAGTGATCGTACAGACGTCCTGCAGCGTCT
GGCAGGTACTGATGTGGACGTCGACGTGCTGGCTGGCCACTTGTTAGAGCCGAT
CCCGCACTGGTTAGGCGAACAACCGTTATTGACGTCCGTGGAGCCCGTGCCACC
GACACATCACGTTATTTTAACTCCGGACGCACGTAGTCGTTTACGGCCATCACG
TCGTCGTCGGCCTGAACCGTCGCTGGGTTTGCGGGACGGTGCGGAACTTGTCCG
GGAGCGTCTGGCCGCAGCTGTGGCTACCCGTGTGGACAGTCCAGCGTTAATTAC
CAGTGAACTGAGTGGCGGCTATGATTCCACTAGTGTGTCATACTTGGCAGCGCG
CGGTAAAGCCGAGGTGGTGCTGGTCACGGCCGCGGGACGTGACAGCACAAGCGA
GGATCTGTGGTGGGCTGAACGCGCAGCCGCAGGGCTCCCGGAACTCGATCACGT
AGTGTTACCTGCGGATGAATTACCGTTTACGTACGCCGGCCTGACGGAGCCTGG
TGCACTTTTGGATGAACCGTGTACGGCTGTTGCCGGCCGTGAGCGTGTACTGGC
GCTGGTACGTAAAGCCGCGGCCCGCGGCTCTACACTTCATCTGACTGGCCATGG
TGGCGATCACCTGTTTACTTCACTGCCGACACCGTTTCATGACCTGTTTCGTAC
GCGTCCAGTCGCCGCGCTCCGCCAGTTGCGTGCATTTGGCGCGTTGGCTGCGTG
GCCGACCCGTAAGCTGATGCGCGAACTCGCGGACCGCCGCGATCATAGCACCTG
GTGGCGCGCGCACGCACGTCCTCAGAATGGCCAGCCGGATCCGCACAGCCCCAT
GTTAGGCTGGGCAATTCCCCCGACTGTCCCGGCGTGGGTTACTGCTGACGGCGT
GCGCGCGATCGAACTTGGGATTTTAGAAATGGCAGAACGCGCGGAGCCCCTTGG
TCATGCGCGCGGAGAACACGCTGAGCTGGATTCAATCTTTGAAGGGGCGCGTAT
GGCCCGTGGCCTCAATCGTATGGCTACGCATGCCGGAGTCCCGCTTGCAGCCCC
GTTCCATGACGATCGGGTCGTGGAAGCGTGTCTGTCGATCCGGCCGGAGGAACG
catttctgcatggcagtacaaacccttactgaacgccgcaatgcagggtgtggt
GCCGAGCACCGTTCTTGATCGTAGCGCTAAAGATGACGGGAGTATTGATGTGGC
CTATGGGCTGCAGGAACACCGTGATGAACTGGTAGCGCTGTGGGAATCATCACG
TCTGGCGGAAACCGGTCTGATTGATGCGGGTATGCTGCGGCGTTTATGCGCGCA
GCCGTCCTCCCACGAGCTCGAGCATGGATCCTTGTACGCTACTATCGCTTGTGA
GTCTTTTACGGCTACGGAATACGGCGGCGTGCTGCTGGATGAAACCAAAGGCGC
ATACTGGCGTCTGAACACCACAGGCGCCGAAGTTGTTCGCGCCATGGGGGAAGC
CGAGCGGGATGAGATTGTACGGCATGTGGTGGCGACCTTCGATGTTGATGCGCA
AACCGCAGCCCAGGATGTCGATGTCCTGCTGGCAGAACTTCGTGATGCCGGCCT
AATATGGCTCTCCGTGGCCATGGTATGTCCGGTCGCCGTCGTCGCTTAGATGCC
ACGCGTGCTCGCCTGGCCGTTGTGGTTGCCCGTGTCCTGAATCTCTTACCGCCG
CGCTTAATCCGTCGTTGTTTGCGTGTACTGAGTCGCGGAGCCCGCCCTGCCTCG
ATTGAGGCAGCAGAAGCTGCTCGTCGTACTGTGGTTGCGGTGAGTCCAGCTGCC
GCCGGTGCGTACGGCTGTTTAATCCGCAGCATTGCCACCACCCTGGTTCTTCGT
TCACGCGGGCAATGGCCAACCTGGTGTGTTGGTGTACGTGCGGAGCCTCCTTTT
GGTGCCCATGCCTGGATTGAAGCAGAGGAGCGGCTGGTGGATGAACCTGGTACT
ATGCATACTTACCGTCGTCTTATCACCGTTGGTCCACTGTCTCGCAAAGTTCGT
TAATAA
TGGCCGATCTGGTCGATCCACTTCCAGGTCACGCACTGCGCGCTGCGGCGACAT
TACGTCTGGCAGATCTGATTGCGGCTGGTGCAGATACTGCACCGGCATTAGCAG
CGGCGGCACGCATTGATGCTGACGCGATCGCGCGTCTTATGCGGTATCTGTGCA
GTCGCGGGATTTTTCAAGCACATGAAGGCCGGTACGCGTTGACTGAATTTAGCG
AATTGCTGCTGGATGAAGATCCATCTGGCCTGCGTAAAACCTTAGATCAGGATA
GCTATGGGGATCGTTTCGACCGCGCGGTTGCGGAACTGGTGGACGTTGTACGGT
CCGGTGAACCTTCTTATCCTCGCCTTTACGGCTCGACGGTTTATGATGACCTGG
CAGCCGATCCTGCCCTCGGCGAGGTGTTCGCGGATGTTCGTGGCTTGCACTCCG
CAGGGTATGGGGAAGATGTCGCGGCAGTGGCGGGTTGGTCCTCATGCCTGCGCG
TTGTCGATCTGGGTGGAGGGACTGGCTCCGTCCTGCTTGCTGTGTTAGAGCGTC
ACCCGTCCCTGTCAGGCGCAGTACTGGATCTGCCATACGTCGCCCCGCAGGCAA
AGAAAGCTCTGCAGGCCTCAGCGTTTGCCCAACGTTGTGAATTTATCAAAGGGA
GCTTCTTCGATCCGTTACCTCCGGCAGACCGTTACCTGTTGTGTAACGTGCTGT
TCAACTGGGATGACGCGCAAGCAGGCGCTATTTTGGCACGCTGTGCGCAGGCGG
GCCCTGTGGCCGGAGTAGTGGTAGCCGAACGTTTGATCGATCCGGATGCGGAAG
TGGAACTCGTAGCAGCTCAAGATCTGCGTCTGTTGGCTGTTTGCGGCGGTCGGC
AGCGTGGCACCGCTGAATTCGAAGCGCTTGGGGCAGCCCATGGCCTGGCGTTAA
CCAGCGTTACCCTCACGGCATCTGGTATGAGCCTGCTCCGTTTCGATGTGTGTC
GTGCCGGGAGTGCTGGCGGGGAAGTTGTGGAAAAATCTTAATAA
GCGACGGCCCCTCGTCACGTGCGTGCCCTGGATTTCGGTCATGTTCTGGTCCTG
ATCGATTACCGTTCCAATCACGTCCAGTGCCTGCTTCCGGCAGCCGCAGCCCAT
TGGACAGCCACAGCGCGTACCGGCCGCTTGGACACCATGCCGGCAGCGCTGGCC
ACCCAGTTACTGACATCGGCGTTATTAGTACCGCGGCCGACCGCAACACCGTGG
ACGGCACCTGTAGCGGCACCACCTGCTCCACCGTCATGGGGTGGATCCGAGCAT
CCTGCCGGGACATCACGCCCTCGGGCACGTCATCGGCACTCAACCACGGCTGCG
GCGGCGCTGGCATGTGTGCTGGCGATTAAGGCAGCAGGCCCAACCCGCTATGCT
ATGCAGCGCTTGACCACGGTCGTGAAGGCAGCCGCTTCTACGTGCCGTCGCCCG
GCAACGCCAGCACAAGCGACGGCTGCTGCGCTTGCGGTCCGTCAGGCATGCTGG
TACTCGCCAGCGCGTACAGCCTGTCTGGAAGAATCCGCCGCGACTGTCATTTTA
CTCGCTACCCGGCGTTTGAGTTCGACATGGTGCCATGGAGTAGCTCCCGATCCG
ATTCGCCTCCATGCCTGGGTGGAAACTGAGGATGGGACACCTGTAGCAGAGCCA
GCCTCGACCCTTGCGTACACCCCGGCCTTAACCATTGGAGGCCACCATCAACAC
GGATTTTCGACGACCCGTGAAGTTCGTCAACGCCCTGGTAATGCCGAGTTTATT
GCTACGGACTCGCCTATTTGGCGCCTCGGTCGTAGTCCAGCTCGTTGCGTGGCT
GCGGACCATGGACAGCGTCGCCTGGTAGTGTTGGGAGAATGCGGGGCAACGGAT
GGCGAATTATCTCGCCTGGCGACCGCGGGGCTGCCCACGGATATTACCTGGCGC
TGGCCAGGCGTGTACGTGGTGGTCGAAGAACAACCGGAACGTACGGTGCTGCAC
ACTGATCCAGCAGCTGCACTCCCGGTATACGCAACCCCTTGGCAAGGCGGCTGG
GCATGGTCAACCAGCGCGCGCATCCTGGCACGTTTAACAGAAGCTCCAATTGAT
GGTCAACGCCTGGCATGTTCAGTGCTGGCCCCGTCTGTTCCGGCTCTGAGCGGT
ACCCGCACATTCTTTGCGGGTATCGAACAATTGGCCCTGGGTTCGCGTATTGAA
CTGCCGGTGGATGGGTCCCGTCTGCGTGTTACGGTACGTTGGCGCCCGGATCCA
GTCCCGGGAGAACCATATCATCGCTTGCGCACAGCGTTGACCGAGGCGGTCGCC
CTGCGTGTCAACCGCGCACCAGACCTGTCATGCGACCTCTCGGGCGGCCTCGAT
TCCACGTCACTGGCAGTCCTGGCGGCTGTGTGCTTACCGGAGTCCCACCATCTG
AATGCTATCACGATTCATCCGGAGGGCGATGAAAGTGGCGCGGACTTACGGTAT
GCGCGCTTGGCAGCTGCGCACCACGGGCGTATTCGCCACCACCTTCTCCCCCTT
GCGGCAGAACACCTGCCGTATACTGAAATTACGGCGGTGCCCCCTACCACCGAA
CCGGCACCTTCAACATTAACGCGTGCACGCCTCGCGTGGCAGTTAGATTGGATG
CGCCAGCACTTAGGCAGCCGCACCCATATGACTGGCGATGGAGGCGACAGCGTA
CTGTTCCAACCGCCGGCACATCTGGCGGATCTCCTGCGGCATCGGCAGTGGCGT
CGGACTTTGTCGGAAAGTTTGGGATGGGCACGCCTTCGCCATACGTCTGTTTTA
CCCTTACTGCGTGGAGCAGCAACTCTTGCACGTACATCACGTCGGTCGGGCCTC
CAGGATCTCGCACGCGCATTGGCGGGTGCAGGTCAGCAGGGCGATGGTCGTGGC
AATGTGAGCTGGTTCGCACCATTACCGCTGCCTGGCTGGGCGACCCCAACCGCT
CGTCGCTTACTGCTTGATGCAGCCGATGAAGCTATCTCGACCGCGGATCCGTTA
CCGGGACTGGATACGTCGCTGCGCGTACTGATCGATGAAATTCGCGAAGTCGCC
CGCACGGCAGCGGCAGATGCCGAACTGGCGGATGCTCACGGAACGACTCTGCAT
AACCCATTTCTCGATCCGCGCACTATTGATGCAGTCCTGCGCACGCCAATCGCA
CATCGCCCGGCGGTCCACTCGTATAAGCCAGCGCTGGGGCATGCAATGCAGGAT
TTGCTCCCGGGTGCAGTCGCTCGGCGCTCAACTAAAGGCTCTTTTAACGCCGAT
CATTATGCGGGGATGCGTGCAAATCTGCCAGCATTGACAGCGCTGGCAGATGGC
CACCTGGCCGACCTGGGTTTGTTGGAGCCGACGCGCTTCCGCAGTCATCTTCGC
CAAGCCGCCGCGGGCATTCCGATGCCGCTTGCGGCGATCGAACAGGCGCTGTCT
GCCGAAGCATGGTGTCATGCACATCACGCCACCCCAAGCCCTGCCTGGACAACG
CAGCCACCGGAACACCCGCATGCCTAATAA
CCCTCTGGATCTCAACTGATACCTGTGGTCTGGGGCCGTATCGCGCTGACTTGG
TGGATACCTATTGGCAGTGGGAACAAGACCCAACATTGCTTGTAGGCTACGGTC
GTCAGTCACCGCAGTCACTGGAGGCCCGCACGGAAGGTATGGCCCACCAATTGC
GTGGCGATAACATCCGTTTCACTATCTATGATCTGTGCAGCAGTACACCTACCC
CGGCGGGCGTGGCAACGCTGCTGCCCGATCATAGCGTCCGTACTGCCGAGTATG
TTATTATGCTTGCGCCTGAAGCACGTGGGCGTGGCTTAGGAACCACCGCCACGC
AGCTGACGTTAGATTATGCGTTTCACATCACCAATCTGCGGATGGTCTGGTTGA
AAGTACTGGCGCCGAACACCGCGGGCATCCGTGCGTATGAGAAAGCTGGCTTTC
GTACAGTTGGAGCGCTTCGCGAAGCCGGCTATTGGCTGGGGAAGGTCTGCGATG
AGGTACTGATGGATGCCTTAGCGAAAGACTTCACGGGTCCAAGTGCAGTCCACG
CAGCATTAACTGGCGCCAGCGGTCGCCAGCTGCGCCGTGCACCTTAATAA
TGGAAGTAACGCATTACACAGATCCTGAAGTTCTGGCCATTGTTAAAGATTTTC
ATGTCAGAGGTAACTTTGCTTCCCTCCCCGAATTTGCTGAACGAACTTTCGTGT
CCGCGGTACCTCTTGCCCATCTGGAGAAATTTGAAAATAAAGAAGTTCTCTTCA
GGCCAGGTTTCAGCTCCGTAATAAACATATCCTCATCACATAATTTTAGTCGTG
AAAGGCTCCCATCAGGAATAAACTTTTGCGACAAAAATAAACTTTCCATTCGTA
CTATTGAAAAGTTATTAGTCAATGCATTCAGCTCACCTGATCCTGGCTCTGTAA
GGCGGCCTTATCCTTCTGGGGGGGCATTGTACCCGATTGAAGTTTTTTTATGCA
GATTATCTGAAAATACAGAAAACTGGCAGGCAGGAACTAATGTTTATCACTACC
TGCCGCTAAGTCAGGCACTAGAACCTGTTGCTACATGTAATACTCAGTCACTCT
ACCGAAGCCTGTCCGGTGGGGATTCGGAACGTCTTGGTAAACCCCATTTTGCTC
TCGTCTATTGCATTATTTTTGAAAAAGCTTTGTTCAAATATCGCTACAGAGGAT
ACCGGATGGCCTTAATGGAAACAGGTTCGATGTATCAGAACGCAGTATTGGTTG
CAGATCAAATAGGACTGAAAAACCGGGTATGGGCGGGATATACCGATTCATACG
TAGCAAAAACAATGAATCTGGATCAGAGGACTGTAGCGCCACTGATCGTTCAGT
TTTTTGGAGATGTAAACGATGATAAATGTCTACAGTAACCTTATGTCCGCATGG
CCGGCCACAATGGCCATGAGTCCAAAACTGAACAGAAATATGCCAACGTTTTCT
CAGATATGGGACTATGAGCGTATTACACCAGCCAGCGCGGCCGGTGAAACTCTG
AAGTCAATTCAGGGGGCAATAGGTGAATATTTTGAACGCCGTCATTTTTTTAAT
GAGATAGTCACCGGTGGTCAGAAAACATTATATGAGATGATGCCTCCATCTGCT
GCAAAGGCTTTTACCGAAGCATTTTTTCAGATCTCATCACTGACCCGCGATGAA
ATCATAACCCATAAATTTAAAACGGTCAGAGCCTTTAATCTGTTTAGCCTTGAA
CAACAAGAAATACCTGCAGTCATAATTGCACTCGACAATATAACCGCTGCAGAT
GATCTGAAATTTTATCCTGACAGAGATACATGCGGATGTAGCTTTCATGGTAGT
TTGAACGATGCCATAGAAGGTTCCTTGTGTGAATTTATGGAGAGACAGTCCCTC
CTTCTTTACTGGTTACAGGGAAAAGCCAATACTGAAATATCCAGTGAAATAGTA
ACAGGCATAAATCATATAGATGAGATTTTACTGGCTCTCAGGTCAGAAGGAGAT
ATCAGGATTTTCGATATCACCCTGCCCGGAGCTCCTGGACACGCAGTACTAACC
CTGTATGGCACAAAAAACAAAATCAGTCGAATAAAATACAGTACCGGATTATCC
TATGCTAATAGTCTGAAAAAAGCACTTTGTAAATCCGTAGTGGAATTGTGGCAA
TCGTATATATGCCTGCACAACTTTCTTATTGGCGGTTATACTGATGATGACATT
ATTGATAGTTACCAGCGTCACTTTATGTCATGCAACAAGTACGAGTCGTTTACG
GATTTGTGTGAAAATACGGTACTACTGTCTGATGATGTCAAGTTAACGTTTGAG
GAAAATATTACGTCAGACACAAATTTATTAAACTATCTTCAACAAATTTCTGAT
AATATTTTTGTTTACTATGCCAGGGAAAGAGTAAGTAACAGCCTTGTCTGGTAC
ACAAAAATAGTAAGCCCTGATTTTTTCCTTCATATGAATAACTCAGGTGCAATA
AACATTAATAATAAAATTTACCATACCGGGGACGGTATTAAAGTCAGAGAATCA
AAGATGGTACCATTCCCATAATAA
ATCCGTATCCAGTGTATCGTCGTCTGCGTGATGAGGCTCCGTGCCACCATGAAC
CAGCGTTAGGTCTGTATGCGTTGAGCCGCTACGAGGACGTTCTGGCTGCCCTTC
GTCAGCCCACCGTGTTCAGCTCAGCAGCGCGTGCGGTAGCCTCCAGTGCAGCGG
GAGCAGGTCCATACCGCGGTGCCGACACCGTTAGTCCGGAGCGGGAAACTGCGG
CTGAAGGGCCCGCCCGTAGCCTGTTGTTCCTGGATCCGCCAGAGCACCAGGTGC
TGCGTCAGGCGGTGTCCCGTGGCTTTACGCCGCAGGCAGTATTGCGCCTTGAGC
CGGCCGTCCGCGACATTGCGGCGGGTCTTGCTGATCGTATCCCCGATCGCGGTG
GTGGCGAGTTCGTTACCGAATTTGCGGCTCCGCTGGCAATCGCAGTGATTCTGC
GGTTACTTGGTGTACCGGAAGCAGATCGTGCCCGCGTAAGCGAACTTTTATCGG
CATCAGCCCTGTCGGGGGCGGAAGCAGAACTGCGCTCCTATTGGCTGGGCCTTT
CGGCACTCCTCCGCGATCGTGAAGATGCAGGCGAAGGTGACGGAGAGGATCGTG
GTGTGGTGGCGGCTCTGGTCCGTCCTGATGCTGGACTGCGCGACGCGGATGTTG
CCGCAGGACCTGCCGTGCGTGCACCGCTGACGGATGAGCAGGTTGCAGCATTCT
GCGCCTTAGTGGGGCAAGCCGGCACTGAAAGTGTGGCAATGGCGCTCTCCAACG
CATTGGTCCTGTTCGGGCGTCACCATGACCAGTGGCGCACACTGTGTGCGCGTC
CGGATGCGATTCCAGCAGCATTCGAAGAGGTCCTCCGCTATTGGGCACCTACGC
AGCATCAAGGTCGGACGTTAACCGCGGCGGTACGTTTACATGGCCGTCTGCTGC
CGGCCGGTGCGCATGTACTGCTGCTGACCGGTTCAGCCGGCCGGGATGAACGTG
CGTACCCAGACCCCGATGTATTTGACATCGGTCGCTTCCACCCGGATCGTCGTC
CGTCGACCGCGCTGGGTTTTGGTCTGGGCGCACACTTTTGTTTAGGCGCTGCTC
TCGCTCGTCTGCAGGCACGCGTAGCGCTGCGCGAACTGACACGCCGGTTCCCGC
GTTATCGTACGGACGAGGAACGCACTGTGCGTTCGGAAGTGATGAACGGGTTCG
GCCACAGCCGTGTACCATTTTCCACGTAATAA
TTCCCAGAGATCAATGAAACGGATTTCGATAACAATATCAAGCCCCTGCTGGAT
GAACTGGAATCTCGTATTACCATTCCGCAGGAGGAACTGAGCTTTTCAAGCATT
AACGATGATTTATTTCGCGAGTTAACCCGCAACGAGGAGTACCCTTACCAGAGC
ATTTGTACGATCGTTGCAAACATCGTGATGGATGACGGCAGTGAGATTTGGCGC
AAAGATATTTTTGTTGATTCCAATAGTGTGCGCGAAGCCGTATGCGACATTCTG
AGCCAAACGTTATTCCTCTATTTCATCCGCTGCTTCTCCGAACAAATTAAAGAC
ATTCGCAAAACTGATGAGGATAAAGAGTCCACCTACAACCGCTACATTAACCTC
CTGTTCAGCTCCAACTTCAAAATCTTCTCCGACGAATACCCTGTCCTGTGGTAT
CGGACCATTCGCATCATCAAAAATCGCTGGTATTCTATCAAGAAATCGTTACTG
CTGACTCAAAAACACCGTGTGGAGATCGATAAGCAGTTGGACATCCCGCACAAG
ATGAAGATTAAAGGCCTGAAAATCGGGGGAGACACGCATAACGGCGGTGCCACA
GTGACCACGATCTTCTTTGAGAAAGGGTATAAACTGATTTATAAGCCGCGGAGC
ACATCCGGCGAATTCTCGTACAAGAAATTTATCGAAAAGATTAACCCGTACCTG
AAGAAAGACATGGGAGCGATTAAAGCGATCGATTTCGGTGAATACGGCTTTTCT
GAGTATATTGAGTGTAACACGGATGAAGAGGACATGAAACAGGTCGGTCAGCTT
GCATTTTTCATGTACCTGTTGAATGCATCAGATATGCATTATAGCAATGTCATT
TGGACCAAACAGGGCCCTGTGCCGATTGATTTAGAAACCTTGTTCCAGCCGGAT
CGTATTCGCAAAGGCCTGAAGCAGTCGGAAACTAACGCGTACCACAAAATGGAG
AAAAGTGTATACGGAACGGGAATTATTCCAATTTCCCTGAGCGTTAAAGGCAAA
AAGGGTGAGGTCGACGTCGGCTTTAGTGGAATCCGTGATGAGCGCTCTAGTTCG
CCGTTTCGCGTTCTGGAAATTTTGGATGGGTTTTCGAGCGACATCAAAATCGTG
TGGAAAAAGCAGCAGAAGTCTAGCTCCAGCAAAAACAATCTGATTGTCGATCAC
AAAAAGGAGCGCGAAATCCTTCAGCGTGCCCAGTCCGTCGTAGAAGGTTTCCAG
GAAACCTCTAAAATCTTCATGAAACATCGTGAGGAATTCATCTCCATTATCTTA
GACTCATTCGAGAACATCAAAATTCGCTACATCCATAACATGACGTTTCGCTAC
GAACAGTTGCTGCGCACTCTGACGGATGCCGAGCCGGCCCAGAAGATTGAGTTA
GACCGTCTGCTGCTGAGTCGTACCGGAATTCTGTCCATCTCGTCTAGTCCCTAC
ATCTCGCTCTCCGAATGTCAACAGATGTGGCAGGGTGACGTGCCGTACTTCTAC
TCGAAGTTTTCGAGCAAAAGTATCTTTGATACCAATGGCTTCGTTGATGAAATC
GAGCTGACGCCCCGCCAGGCATTTATCATCAAAGCCGAAAGTATCACCAACGAT
GAAGTCGATTTTCAGTCCAAGATCATTAAACTGGCGTTCATGGCACGCTTAAGT
GACCCGCACACAACCAACGACAACAAACTGAATAAAAAGGTGATTATCGAAAGC
AACCAGCAGAGCAACAGCAGTGAATCAGGTAACAAAGCCATTTTGTTCCTGAGC
GATCTGCTGAAAAATAACGTACTGGAAGATCGTTATAGTCATCTGCCGAAAACT
TGGATTGGCCCTGTAGCACGTGATGGCGGTTTGGGTTGGGCGCCGGGCGTGCTG
GGATACGATCTGTACTCGGGCCGTACAGGACCTGCGTTAGCATTGGCTGCGGCC
GGGCGCGTTTTGAAAGATAAAGACAGTATCGAACTTAGCGCCGACATTTTTAAT
AAATCGTCCCAGATTCTGCAGGAAAAGACTTACGACTTTCGTAACCTGTTCGCA
TCAGGTATCGGCGGTTTTAGCGGGATTACCGGTCTGTTTTGGGCGCTGAACGCG
GCAGGGAATATTCTGAACAATGATGACTGGATTAAAACCTCGAATCAGAGTATG
CTGCTGCTGAATGAGAACATGCTGAAAGTGGACAAAAATTTCTTTGACCTGATT
AGCGGCAACTCGGGAGCGATCGGTATGATGTACCTGACCAATCCAAATTTCTAT
TTGTCTCGCTCGAAAATTAACGACATTCTGCTGACCACGGACTGCTTGATTACT
GAAATGGAAAAAGACGAAACGAGCGGACTGGCCCATGGCGTGTCTCAGATCCTG
TGGTTCCTTAGCATTATGATGCAACGTCAGCCCTCAAGTGAAATCAAAATCCGC
GCGACGATTGTCGACAACATCATCAAGAAGAAGTATACGAATTCCTATGGCGAA
ATCGAATGCTACTATCCGACTGATGGGCACTCCAAATCCACCTCGTGGTGCAAC
GGGACAAGTGGGATTCTGGTCGCCTATATTGAGGGGTATAAAGCTAATATCGTG
GACAAATCCTCGGTGTATCATATTATTAATCAGATCAACGTCGAACAACTTCAG
CATGATAACATTCCGATCATGTGCCATGGTAGCCTTGGTGTGTATGAATCGCTT
AAATATGCGTCAAAGTACTTTGAAATCGAAACCAAGTACCTTCTGGATGTGATG
CGCAATGGCGGCTGCTCCTCCCAAGAAGTATTAAAGTACTATGGCAAGGGTAAC
GGCCGTTACCCGCTGTCACCAGGTTTAATGGCGGGTCAGTCGGGCGCGTTGCTG
CACTGTTGCAAACTGGAGGATAACGATATCAGCGTGAGCCCCATTTCACTGATG
ACGTAATAA
CGTGGATTCTATCATCGAATTCTACAAAAAGGACATCTACCTGGCATACAAAGA
GCTGGAACGCGAAATCAAAAACATCGATAAGACCATCTACAACACTTCAAATGA
CGAGATCTTGCGGATTTTTAAAGAGAGCCTGATCAGCATCATCACCGATGATAT
TTACCGCCTCTCGATTAAAACCTTCATCTATGAGTTTCACAAGTTTCGTATCGA
TAACGGGTTTCCGGCTGTCAAAGATAGCGAAAGCGCCTTCAATTATTACATCAG
TACCTTTGACGTGAAAACGATCGCTCGCTGGTTTGAGAAATTCCCAATGCTGGA
ATCCATCATCTCCAGTAGCATCAAAAACGATTGCACATTTATGGTGGATGTATG
TGTCAATTTCATCTTAGACCTGTCGGAATGCGAGAAGATTAATCTGATCTCAGA
GGATAGCCGGCTCATCACGATCTCATCCAGCAACTCTGACCCGCACAACGGTGG
CACGCGTGTCTTGTTCTTTCGTTTCCACAACGGTGATACCATTCTTTACAAACC
CCGCAGCCTGACCGTGGACAAGCTGATCTCTAATATTTTCGAAGAGGTATTCGA
ATTCGATGCGACGAACTCGAAAAATCCTATTCCCAAGGTGCTGGATCGGGGTAC
CTATGGCTGGCAGGAATTCATTGAGAAGAAATCGATCTCTTCCTCAGAGATTAA
GCAGGCCTACTATAACCTGGGTATCTTTAGCAGTATCTTTACAGTGTTAGGGTC
TACTGATATCCACGATGAAAACTTGATTTTTAAAGGTACGACCCCGTATTTCAT
CGATCTGGAAACAGCCCTCTCTCCGCGTATCCGGTATGAAGGTAATGAGGAAAA
CCTGTTCTATCGGATGAGCTCATCGTTGTTCACTTCTATCGTGGGGACGACTAT
TATTCCTGCAAAACTTGCTGTCCATTCCCAGGAAATTATGATCGGCGCAATTAA
CACCCCTGCGAAACAGAAAACCAAGAAGGATGGCTTTAACATCATCAACTTCGG
CACGGATGCCGTCGATATCGCAAAACAGAATATTGAGGTGGAGCGTATTGCTAA
CCCTATGCGCATTAAAAATAACATCGTGAACGATCCGCTGCCGTACCAGAACAT
CTTTACGCGCGGCTTCAAAGAGGGGATCAAATCCATCATCCTGAAGAAAGGCTC
GATCATTTCCATTCTGAACAACTTCAACAGCCCGATTCGTTACATCATGCGGCC
GACGGCAAAATATTATTTGATTCTGGATGCCGCGGTATTTCCCGAAAACCTGTA
TTCGGAACAGACACTGAACAAAACCCTGAATTACTTAAAGCCGCCAAAAATCGT
GGAAAATTCCCTGATTTCTAAACAGCTCTTTCTTGCCGAAAAACGCATTCTGTC
CGAAGGCGATATTCCGAGCTTCTATGTGCTGGGCAAAGAGAAAAATATCCGTGC
GCAGAACTTCATTAGCGAACAGATCTTCGAGGAAACCGCGGTCGATAACGCGAT
TCAAATTCTGGAATCCATTTCGCAAGACTGGGTGAATTTTAATGAGCGCCTGAT
TGCGGAGGGCTTCTCCTATATTCGTGAACAGAGTCGTGGCTATCTGTCCAGTGA
TTTTGAGAACTCTGATATTTTCAAAAGCTCACTGACCGAAACAAAGAAGTCCGG
TTATACCGCAATGCTGAAAACAATTATCTCCATGTCGGTCAAGACCTCGGAAAA
CAAAAAGATCGGTTGGCTGCCAGGCATTTATGATGATTATCCGATCAGCTATAT
GAGTGCCGCGTTTTGTTCGTTCCATGATTCCGGCGGTATCATCACTTTGCTTGA
ACACCACTTTGGGCACTGCTCCCCCGAATATAACGAGATGAAGCGCGGGCTGCT
GGAACTGGGCAAAATGTTGAAAATTAACAATAGTAACCTGAGCATCATCTCCGG
CTCAGAGTCTCTGGAATTTCTGTATACGCACCGCGAAGTCGAATGCCTGGAACT
GGAATACATTTTAAACAATTCAGCGGAAATCATGGGCGACGTGTTCCTGGGGAA
ATTAGGCCTTTATCTTATCCTGGCGAGCTACCTGAAAACAGACCTGAAAATTTT
CCAAGATTTCAGTATCATCTGCCAGAAAAACCTCGAGTTTAAAAAGTTCGGGAT
CGCGCACGGTGAATTAGGGTATCTGTGGACCATCTTCCGTATTCAAAACAAACT
GAAGAACAAAAATGCGTGTCTGAGCATCTATCATGAAGTGTTGAACATTTATAA
AGGTAAGCGCATTGAATCCGTGGGATGGTGCAACGGTTTATCGGGTATTCTGAT
GATTTTGTCAGAAATGAGCACCGTATTAGAGAAAAATCAAGACTATCTGTTCAA
GCTGGCAAATCTGAGCACTAAACTGAATGAGGAATCCGTTGACCTGAGTGTGTG
CCACGGCGCCAGCGGGGTGCTTCAAACACTGCTTTTCGTCTATAGCAACACGAA
CGATAAACGTTATCTCAGCCTGGCCAATAAGTATTGGAAGAAAGTGCTGGATAA
CAGCATTAAGTACGGTTTCTACAATGGAGAACGCGATAAGGATTATCTGTTGGG
ATATTTCCAGGGTTGGTCAGGCTTCACGGACAGCGCACTCCTGCTGGATAAATA
CAATAACAATGAGCAAGTGTGGATTCCGATCAACCTGAGCTCCGATATCTATCA
GCATAATCTGAACAACTGCAAAGAGAAGAATTATGAGGGCGATGGCTGCCATAA
ATCTTAATAA
AAAACTAAAACCATTAACGAAAAGATTAAAATTTTCACCAAAGAAGAGGTGATT
GATATCAGTTACTTTGAAGAATGGCGCAGCGTTCGTACTCTGCTTAACGAAAAC
TACTTTAAAATTATGCTCGAGGAAATGAATATTTCCAAAAACCAATTTTCGTAT
GCGCTGCAACCGTTAAACGACGAGTTCAAACTGCATACTAACGTTAAAAATGAA
GAATGGATCAAATGCTTTAATCGCGTCATTAACAATTTTAACTATAAAAATATT
AACTATAAAGTTGGTTTGTACCTGCCTATTCAGCCTTTCTCCGTTTATTTACAG
GAGAAACTGAAAGAGATCCTGAAGAAGCTGAACAACATTAAGATTAATGATAAA
ATTATCGACGCCTTTATCGAAGCTCACCTGATCGAAATGTTCGACCTCGTCGGT
AAAGTAATCGCCCTTAAATTTGAAGATTATAAACAGATCAACTTCCTGAAAAAC
ACAAATAATGGCACCCGCTTGGAGGAATTCTTGCGTAGCACCTTTTATTCTCGG
AAGTCATTTCTGAAACTGTTTAACGAGTTTCCGGTACTCGCGCGGGTTTGCACC
GTACGTACGATCTATTTGATCAATAACTTTAGTGCTATCATCCAGAACATCAAT
AGCGACTACCTGGAAATCCAGGAATTTCTGAACGTCGATTTCCTGAACTTGACA
AACATCACTCTTTCGACGGGTGATTCCCACGAACAGGGTAAAAGTGTGTCCATC
CTCTATTTTGATGAAAAAAAGCTGATTTATAAACCGAAAAATCTGAAGATTTCA
GAAATTTTCGAGAGCTTCATCGACTGGTACACCAACGTCTCTAACCATAAGCTG
CTCGACCTGAAAATCCCGAAAGGAATTTTTAAAGACGATTACACTTATAACGAA
TTTATTGAGCCAAACTACTGCGAGAATAAGCGCGAAATTGAAAATTACTATAAC
CGTTATGGGTACCTGATCGCAATCTGTTATCTGTTCAACCTGAATGACCTGCAT
GTAGAAAATGTGATCGCCCATGGCGAGTACCCGGTTATTGTTGATATTGAAACG
AGCTTTCAAGTCCCTGTGCAAATGGAGGACGATACTTTATATGTGAAGCTGTTG
CGCGAGCTGGAATTGGAAAGCGTTTCATCGTCGTTTCTGTTACCTACCAATCTG
TCGTTTGGTATGGACGATAAAGTGGACCTGTCCGCGCTGAGCGGAACCATGGTC
GAGCTGAATCAGCAAATTCTGGCGCCTGTCAACATTAATATGGACAACTTTCAT
TACGAGAAATCACCGAGCTATTTTCCAGGCGGAAACAATATCCCTAAAAACAAC
AAATCAGTGACTGTTGATTATAAAAAATACTTGCTCAATATTGTGACTGGTTTC
GACGAATTTATGAAGTATACCCAAGAAAATCAGCTGGAATTTATTGAGTTCCTG
AAAAAATTCTCAGATAAAAAAATCCGGGTGCTGGTGAAGGGTACGGAAAAATAT
GCGTCCATGATTCGCTACAGCAACCATCCGAACTACAACAAAGAAATGAAATAT
CGCGAGCGTCTCATGATGAACTTGTGGGCGTACCCTTACAAAGACAAGCGTATT
GTTAATAGCGAAGTACAGGACCTGTTATTTAACGATATCCCGATCTTTTACTCC
TTTCCAAATAGCCGTGACCTCATTGATAGTCGCGGCTTGGTGTATAAAGATTAC
CTTCCTGTGACAGGACTGCAGAAAGCAATTGATCGCGTGAAAGATACCTCGGTA
AAAAGCTTGTTCGACCAGAAGCTGATTCTTCAGAGTAGCTTAGGTCTGTGGGAT
GAGATTCTCAACAAGCCGGTCCAGAAAAAGGAACTGCTCTTTGAAAAGCAGAAC
TTTAACTATGTGAAAGAGGCGATCAATATTGCGGAATTGCTGATTGGCTATTTA
ATCGAAACGGACGACCAGAGCACCATGCTGAGCATTGATTGTTCTGAAGATAAA
CACTGGAAGATTGTTCCTTTAGACGAATCCCTGTATGGTGGGCTGTCCGGCATT
GCATTATTTTTTCTCGATATTTATAAAATTACCAAAGATGAAAAATATTTTAAT
TACTATGATAAAATCATTTCCACGGCCATTAAACAATGTAAAGCGACCATCTTC
TCGTCAAGCTTCACGGGTTGGCTGAGTCCCATTTATCCGTTGATTCTGGAAAAG
AAATACTTTGGTACCATGAAAGATAAGAAATTCTTTGACTACACGATGGAAAAG
CTGTCGAATATGACTGAAGAACAAATTAACAACATGGATGGTATGGACTATATC
AGTGGCAAGGCGGGTATTGTCAAACTGCTGATTAGCGCGTACCGGGAATCGAAG
AACAATGAAAACATCGGACTGGCCCTGAGTAAATTCAGCAACGATCTGATTCAA
AATATTGGCACCGGCAAAGTCAGTGAATTACAAAACGTGGGCCTGGCGCACGGC
ATTTCTGGTATTATGGTCGTAGTAGCCTCACTGGACACGTTTAAAAGTGAATAT
ATTCGCGAGCAGCTGGCAATTGAATATGAGATGTTCTGTTTGCGTGAAGATTCA
TACAAATGGTGTTGGGGCATCTCTGGAATGATTCAAGCCCGTCTCGAAATTCTG
AAACTGAGCCCGGAGTGTGTGGATAAAAAAGAGCTGAACTTGCTTATTAAGCGT
TTTAAAAACATCTTGAATCAGATGATTAACGAAGATTCCCTTTGTCACGGCAAC
GGTTCGATCATTACTACGATGAAGATGATCTATATGTACACCCAAGACACCGAG
TGGAACTCTCTGATTAATCTGTGGTTATCAAATGTAAGTATCTATTCGACCTTA
CAAGGCTATAGCATTCCAAAGCTGGGCGATGTAACAATTAAGGGGTTGTTTGAT
GGCATTTGTGGTATTGGCTGGTTATACCTGTATTCGAACTTTAGCATTGAAAAC
GTGCTGCTCCTCGAGGTCTAATAA
GAGGCCATTAAAGGTTTGACCGTATCAGAACGTTATGACACTCTGAAAAATTCG
GGAGTCAACCTGAATCTGAACATTTCGGCTTTGGAAGAGTGGCGCAACCGTAAG
AATCTTTTAGCCGATGAGGACTTTACGGAGATGCTGACGGTGCTGGAATATGAC
CCGGTGTATTTTAGCCACGCGATTAACGAGAACATCGAAGAACATATCGATATC
TACAAGAGCAAAATTCTGGGGGAAAACTGGTTTATCGTGCTGAACGATATTCTG
GACGAGCTCGATAATCCCATCGAATACAAGAAAGAGATGAATCACAGCTACCTC
CTGCGTCCGTTCTTGCTCTACGCCGAAAAGGAGATGAACAAATACATTGTCAAT
CGTAAGGAGTTACTTCCGGTGGAACCCCAGGTCATCCAACAGATCATGGAAAAT
TTGGCCTCCAAACTGTTCGCCGTTTCTGTGAAAAGCTTTGTCCTGGAGCTGAAT
ATTTCGAAATTGAAGGACGAACTGGCCGGCGAAACACCGGACGAACGCTTTCAC
TCATTTATTCGTTTGATGGGTGAGAAAACGCGCCTGGTGGACTTTTACAACGAA
TATATCGTTCTGAGTCGTATTCTGGTGAACATCACGATCTTATTCGTCAACAAC
ATTATTGAGCTGTTTGAGCGCCTGCAGGAATCCAAGCTGGATATTGTTAAGAAA
CTTGGCGTGCAGGAGGAGTTCAAAATCAGTAATATTAGCATTGGCGAAGGTGAT
ACACATCAGCAAGGACGCTCGGTTATCGTTCTTACGTTCGTGAGTGGAAAGAAA
GTGGTGTATAAACCAAAAAATCTGAAAGTTGTTTCTGCTTATAATTCTTTAATT
GACTGGATCAACAATAAAAATAATATTCTGAAAATGCCTTCGTATAACACATTG
ATTTATGATGATTTCGTGATCGAGGAGTTTGTCGAGAAACGTGACTGCAAAAGT
ATCGAGGAGGTCAAAAAATATTATATTCGTTATGGGCAAATTTTGGGGATTATG
TATATCTTAAATGGGAACGATTTTCATATGGAAAACCTGATTGCCTCGGGTGAA
TATCCGATCATTGTTGACTTGGAAACGCTGCTTCAGAACATTATCAATTTTAAA
AACAAACCATCAGCGGACTTGATCACCACCAAAAAGATGCTTAACCTGGTAAAC
AGTACTCTGCTGCTCCCTGAAAAACTTCTGAAGGGCGACATCACGGACGAAGGA
ATCGACATGTCAGCCTTGGCAGGGAAAGAACAACACTTGGAACGCCGCGAATAC
CAGTTGAAAAACCTGTTCACCGACAACATGGTTTTTGATCTCGAAAAAGTGAAA
ATCGAAGGTGCGAACAACATCCCGAAATTAAACGGTGAAAACGTTGACTACAGC
ACCTATATTGATGAGATTGTGGTTGGGTTCGAAAATATCTGTAACCTGTTCATT
CAATATCGCGACGAGTTACTGCATTCCGGCATCCTGGAGGAGTTTAAAGATGTG
AAGGTTCGTCATGTGCTTCGCAATACGGTTGTTTATGCTAAGATGCTGGCGAAT
ACATATCATCCAGATTACCTGCGTGATTCGTTGAATCGCGAACAGGTTCTTGAA
AACATTTGGGTGCATCCGTTTGAGCGCAAAGAATTCATTAAGAGCGAGATGGAA
GATATCCTCAACAACGACATCCCGATCTTTTTCTCATACGCGTCGTCTAAGGAT
ATTATCGATTCGAATGGCAAACTGCACAAAAACGTTATGGAAATTTCGGGTTAC
GAACGTTTTACCACCAAACTGAAGGAACTGAATCCCTTTCTGATTGAACAGCAG
GTGAGCGTTATTAATATTAAAACCGGCCGCTATGGGGATAAGAAATTCGAAAAA
AATTATAGCGTGCGCGACGTTGCAACGGAGAAAAAAGATAATCCGATTGATTTC
CTGCAGGAGGCAATGAATATCGGCGATAAAATTTTGGAACATGCTATCATCTGT
GATGAGACCAAAACGATTTCGTGGCTTACCATTAACAACCATCATGATAAAAAT
TGGGAAATTGGGCCTATTTCCGGTGAATTTTATGATGGTCTGGCGGGAATTTCA
CTCTTCTACCACTACCTCTATAAAAAATCCCACAATGTCGAGTATAAAAAAATT
CGTGATTACGCGTTCAACATGGCGAAAGTCAAAGCCCTGTCACTGAAATACGAT
AGTGGCTTGACCGGTTACGCTTCCTTGCTGTATACGGCACACAAGATTGTTCAG
GATGAACCGCGGAAGCAATACAAAGACGTGATCAACGAAGTGTTCAAGTACATT
GATGAGAGCAAAGTCGTGACCGCTAAGTATAACTGGTTGCATGGCACTGCCTCT
ATTATTCATGTGTTATTGAACCTCTACGAGGACTCTCGTGATATGGCGTACCTG
ACTAAATGTATTCAGTACGGCAAATATTTGGTCAAGCAAATCAAAGAACACAAG
GATATGCTTGCGCCTGGCTTTAGCCAGGGCATCTCTTCGGTCATTATGGTTCTG
GTGCGCTTAAGTAAAAAGTGTGAAGTCGAAGAATTTCTCGAATTAGCTCTGGAA
TTAATGGAAATGGAACGCAACAAACTGGGAAACCTTTCTGAATCAAACTGGCTG
AACGGCTTGGTGGGCATTGGCTTATCACGTATCAAACTGAAAGGACTGGATTCC
AACTTACAGGTCGACAACGACATCGAACTCGTCCTGGATGGCGTCATGAACAGC
TTGTACTCAAAAGATGATACTTTGAGCTGTGGTAACTCTGGCACAGTGGAATTG
TTCCTGAGTCTGTTTGAACAGACGAAAAAGAAAGAGTATCTGGATATGGCGAAA
GCAATCTGCGGGAAAATGATCGAAGAGAGTCGCATCTCCTTTGAGTATCAGACA
AAGAGTCTGCCGGGTTTAGAACTGGTGGGCCTCTACTCTGGCTTAGCCGGAATT
GGTTATCAATTCTTACGTATCTCGGACGTTGAGGATATTGCGAGCATTGCTACC
TTAGATTAATAA
TAGGAAGTCCGGATGATCTTCACGTCCAGTCAGTGACGGAGGGTCTGCGTGCAC
GCGGTCACGAGCCTTACGTGTTTGACACCCAACGTTTTCCGGAAGAGATGACAG
TGTCACTTGGTGAACAGGGTGCCTCTATTTTTGTCGATGGCCAGCAAATTGCAC
GTCCGGCGGCGGTGTACCTCCGTTCACTGTACCAGAGCCCCGGCGCGTATGGGG
TGGATGCCGACAAAGCGATGCAGGATAACTGGCGCCGCACATTGCTCGCTTTTC
GCGAGCGTAGTACCCTGATGAGCGCTGTGCTTCTGCGTTGGGAAGAAGCGGGGA
CTGCAGTGTATAATTCGCCACGCGCGTCGGCGAATATCACTAAACCGTTTCAGC
TGGCGCTGCTGCGCGACGCTGGTCTGCCGGTACCACGTAGCTTGTGGACAAACG
ACCCTGAAGCAGTGCGGCGGTTTCATGCGGAAGTGGGTGACTGTATTTACAAAC
CGGTCGCCGGGGGAGCGCGTACACGCAAACTGGAAGCGAAAGATCTCGAAGCGG
ACCGCATCGAACGCCTGAGTGCAGCGCCGGTGTGTTTTCAAGAACTGCTCACAG
GAGATGATGTGCGTGTTTACGTGATAGATGACCAGGTAATATGCGCCCTGCGCA
TCGTAACTGATGAGATCGATTTCCGCCAAGCAGAGGAACGTATCGAGGCCATCG
AAATTTCAGATGAAGTAAAAGACCAATGTGTACGTGCCGCCAAACTTGTTGGCC
TGCGCTACACCGGTATGGATATCAAAGCCGGCGCCGATGGTAACTATCGTGTTC
TCGAACTGAACGCGAGTGCGATGTTTCGCGGTTTCGAAGGCCGTGCGAATGTGG
ATATCTGTGGACCGCTGTGTGATGCATTGATCGCTCAGACCAAACGTTAATAA
CCACGATAACGAGAGCATTTCATTGGTAACCCAAGCCATTGAATCCCAGGGTGG
TAAAGCATTTCGCTTCGATACCGATCGTTTTCCGACGGAAGTCCAGCTGGACAT
CTATTACTCAAATACAGAGAAATGCGTGCTGGTGGCTGACGATCAAAAACTGGA
TTTAAATGAAGTAACCGCGGTCTGGTATCGCCGCATTGCGATCGGTGGCAAAAT
CCCGCCCACGATGGATAAGCAACTTCGTCAGGCCTCGATTCAGGAGAGTCGTGC
TACAATTCAAGGCATGATAGCGAGCATTCGCGGCTTTCACCTTGACCCAGTGCC
GAACATTCGTCGCGCTGAAAATAAGCAACTGCAGCTGCAGGTTGCCCGCAAAAT
CGGACTGGATACCCCACGCACTCTCACCACTAATAATCCGCAGGCCGTGAAGGA
ATTTGCGGCAGAATGCCAGCAGGACGTAATCACCAAAATGCTGAGTAGTTTTGC
GATTTATGATGAGAAAGGCGGAGAACAGGTGGTTTTCACCAATCCCGTGAAATC
TGAGGATCTGGAAAATTTAGAAGGTCTGCGCTTTTGCCCTATGACGTTTCAAGA
GAAAATCGCAAAGGTTCTGGAGCTCCGGATCACCATCGTGGGTAAGTCAATTTT
AACGGCTGCGGTGAATTCACAGGCCCTGGACAAATCCCGTTATGATTGGCGCAA
GCAGGGCGTAGCATTACTGGATGCATGGCAGACCCATACGTTACCCCAGGACGT
GGCTGATAAATTGCTTCAACTGATGGCCCATTTCGGGTTAAACTATGGAGCCAT
TGACGTGATTCTGACCCCGGATAATCGCTATGTGTTCTTGGAGGTCAATCCGGT
GGGCGAATTCTTTTGGCTTGAGCGTTGCCCAGGTCTGCCGATTAGTCAAGCTAT
TGCTAAAGTGCTGCTTTCTCATATATAATAA
AGGTTTGGCCCTCGTGGATCAGCATCCGATTTTTCTGGACCTGAAAACAGACCG
TTACCTGTCGTTGAGTCCAGATGGGGCAGCAGTCCTGCTGGGAGCAGCGCCAGC
CACCAAAGAGAGTCCACTGTTTCTCGGATTAGAATCCATTGGCTTGGTCAAAAA
CGGTCCGTCAGGCCTTAAGCCTTGCCAAATTGCCGTAGCCACTGGGTCTGCACC
GCCCCGTAAGGTGCAATTCGAGTCGTTGTCACTCCTGCTTTTGCGCTTAATTCG
TGCACGTCTGGATCAACGTGCTCTTTTGAAGCGTGTGACCGACTTAAAGAAGGC
CGGCACCATTGCCCAGACGAAGAACCGTGACTGCGCCTTGTCATTATTAGGTAG
CGTGGAGACTGAGGCAAAGGCTTGTCGTACCCTTTTAAGTAGTACAGACAAATG
CCTGCCCGACGCATTCGCAATTGCAACGCACCTGCGCCGTCGCGGAGTAGACGC
CAAGTTAGTTTTCGGTGTGCGCCTGCCATTCGCGGCACATGCCTGGGTCCAGGT
AGATGATATTGTAGTGGGTGATCGTCCCGACCGTATCCTTGCGTTCACCCCCAT
TATGTCGCGTCTTTCTTTGTTCGCGGACATGTCAGCACACCAGCACTGCGTCAC
CCAGAGCCAAAGGGTTTCGCTTATGCAAAAGTCAGTGGCGGACTGAGCGTATGG
AGCGATGCGCCGATTCGTCACCGTGCGCCCCTTATTACAGTGGGCGCGGTGTTC
GATCGCGCGTCTTTTAAAGGGCTGGATTGCGACTTATCAGGTCTGCGTCAGGAT
GGTCTTAATACATTGAAAGCGGAAACGTTCGGACCCTACCTGGCGTTAGAGGTT
GCCGATAACGGCACCCTTCGCGTTTATCGCGATCCGTCAGGCGGCGCGCCTTGC
TATTACCTGCAGACCGAGGACGGCTTCTGGCTTGCAAGCGATGCTGATTTGTTA
TTCACTCATTCGGGCGTACATCCATCAGTAAGCTTACCGGGACTGATTGAACAC
TTGCGTCGTCCAGAGTTCCAAAATGAGGGCACATGCTTAAACGTCAAGCAAGTA
CGCCCTGGGGAGCAGGTTGATTTATCGCTCTCGGGCGAGGTCCGTGCCTGTTTG
TTCCCGCCTGCATCATCCCTGCGCCCGCCTGAGTTGCACCGCGCATACGATGAC
ATTAAGGCTGAGCTGCGCGCTCTGATTTTACGCAGCATTAAGGCCTATGCCAGT
GATTTCCCTCACGTTGTTGTTAGCTTCAGCGGTGGTCTGGATAGCAGTGTTGTT
GCGGCCGGCTTAGCGCAAACTTCCACTAAGGTCCTGCTTCACACCTTTAAGGGC
CCAGATGCCAAAGGGGACGAGACTGCCTTCGCCGCAGAATGCGCGGCATATCTG
GGTTTAAGCTTAGAGATTGATACTCTCAGTATCGATGACGTTGATCTGTCGGCA
ACTATTTCCCCGCACCTGCCGCGCCCCAGCACATCATTCTTCTTGCCATCACTG
CTGCGCGGTTTCTCTACCTCGAGCCAAACGCGCACAGGCGGGGCAATCTTTTCG
GGAAACGGCGGTGACTCGGTCTTTTGTTTCATGCATAGCGCGACCCCGCTGGCC
GATTTGATGTGTCGTCCGTCAGGTCTTACGCCGTTCATGCAAACATGGGCCGAC
GTGCAAAAGCTTACCCGTGCCTCAGCGACCGAAGTGCTGCGTCGCGCGTTAAAG
ACAGCCATGGCGCGTGGCTACATCTGGCCTGAATCCAATCTCCTCTTGTCCCGC
GACACAAGCTCGAGCCGTTTAACACCTGACTCCGTTCTGTCGAGCCTTGAGGGG
ATTCTGCCCGGTCGCTTGCGTCACCTCGCCCTGATTCGTCGTGCTCACAACACC
TTCGAGCCATTCGCCCCTTGGCGTACGCCGCCAGTCGTTCACCCTCTCATGGCC
AAGCCGATTCAAGCCTTCTGCCTTTCTCTTCCTTCATGGATGTGGGTCAGCGGT
GGTAAAGACCGCTCGCTCGTGCGTGACGCGTTCGAAGGATTACTTCCAGATTCA
GTGCGCCTTCGTAAATCAAAGGGAAGTCCTGCAGGCTTTCTGCATGCGCTGTAC
CGCGCCAAGGGTCGTCAAATGATTGAGCGTATCCGTCACGGTTACCTGCGTCGT
GAGGGGATCATCGATATCTCTACTGGCCCGGACGCATTGTTCTCGGAAGGGTTC
CGCAATCCGCGTGTAATGCACCGTTTCTTTGAGCTCGCCGCAACTGAGGTGTGG
ATCGATCACTGGCGCAACTGGCGCCGCCCCCGCACATAATAA
CCACGCGGTCGCTCTGGACGAAGATATCGTGGTGCTGGATGCGGTGAGCGACGC
ATACCTGTGTTTAGTTGGTGCCAGCGCTCTGATCAGCTTGGGCAGCGAGCGTTC
CGTCAGTGCAGATCCGGTGGCCGCTGAGACACTTCGTGAGGCTGGTCTGGTGGG
TCCACATCCTAGCGGCGCCACCCGACCAATACCTCCGAAGCCGACGATTGACTT
ACCTGATGCAGCCCGTCAGGCGCAAGGTCGTGAATTACGTGCCGCCGCGTGGGC
TGGCGCGGCAACCGCAATCGATTTCCGCCGGCGTTCATTTAGACAACTCCTCGC
GAGAGCAGGGCAACGCCCGCCGGGTCAAGCAGCTGCTCCGGCTGATGAGGTATT
GGCAGCAGCCGCAGTGTTCATGCGGTTACGTCCATGGTCACCCGTTGGAGGCGC
GTGCCTTATGCGTTCGTATTACTTATTACGGCATTTGCGCATCCTCGGTTTCGA
TGCCGATTGGATCATTGGTGTGCGTACGTGGCCATTTATGGCCCATTGCTGGCT
GCAGGTCGGTGCCGTCGCACTCGACGATGACGTCGAGAGATTAACAGCATACAC
ACCTGGCTCTGTACTGGCCGCGCGGCATGCCCGGTGTAGCTGCAGACGCAATGC
GGGCCGCCATCGAAGCTGAGGGCGCCTGGACCCTGGCGTTCGAGGCCTACCAGC
TGGTAGTGTATGTCAAAGGGCCCCGAGCACCTAAAGTGCGTGCCCTGCCGGATC
AGGGCGGGGTGGTCATTGGGGAACTGTTTGATACTGCAGCAACCCGCGAAGGAC
GCGTGCAGGACTTTCCTATAGCGCTGATCAAAGACGTCGCAGCTCAGGATGCCG
CACGTATTCTTGCTACCCATGCGTGGGGTCGTTATGTGGCTGTATTAAAAGCCG
GTGATCGTCCGCCATGGATCTTTCGCGATCCAAGCGGGGCGGTGGAATGTCTGG
CGTGGGTCCGCGATGAAGTGACCATCATTAGCAGCGATGTTGCAGCGCAACGAG
CTTGGTCCCCTGATCGGCTGGCGATTGACTGGTCGGGACTGGGACGTGTACTGG
CACGCGGAAACTTATGGGGAGAAATTTGCCCGCTGGCTGGCGTCACGGCGATTG
CGCCAGGTACCGCACGGTGTGATCTCGGTGATGCAGCTCTGAGCCTGTGGCGCC
CAGGAGATCATGCACGTCGTAGTCGTCATGATGTTTCCCCACGTGATTTGGCAA
GAGTGGTGGATGCTAGCGTTGCAGCCCTGGCTAGAGATCGCAGCGCTATTCTGG
TCGAAATCAGCGGGGGACTGGATTCCGCTATCGTTGCCACGTCGCTGGCTCGTT
GTGGAGCCCCAGTTGTTGCTGGAATTAACCATTACTGGCCCGAACCGGAGGGTG
ATGAACGTCGCTGGGCCCAGGACATCGCAGATCGGTGCGGTTTTCGCCTGATCG
CGGGCCAACGTCAGCGGCTGTTGCTGGACGAGGCAAAGCTGCTGAGACATGCAC
AGGGCCCGCGACCTGGTCTGAATGCGCAGGACCCGGACCTCGATCACGATCTGG
CGGAACAGGCTAAAGCGTTGGGTGCCGATGCACTGTTCTCAGGGCAAGGTGGCG
ATGGTGTGTTCTATCAAATGGCAAATGCTGCACTGGCAGCCGATATCCTCATGG
GGAAACCTGCTCCTATGGGTAGAGCCGCGTCTTTAGCCGCTGTGGCTCGTCGGG
CACGAGCCACGGTCTGGAGTTTGTGCGGCCAGGCTATGTTTCCGTCGCGCGCAT
TTGCCGCTGGTATGCCGCCGCCAAGTTTCTTGAGCGCCGGTTTGGCGCCGCCAC
CCGTGCACCCGTGGATTGCAGACCAGCGCGGTGTTTCACCGGCGAAACGTATTC
AAATTCGGGGGCTGACCAATATTCAATGTGCTTTCGGCGATAGCTTACGGGGCC
GAGCAGCAGATCTTTTATATCCGCTTATGGCCCAACCGGTCATGGAACTGTGTC
TGTCTATCCCTGCACCGCTGTTGGCAGTAGGCGCATTGGATCGCCCTTTCGCAC
GTGCGGCGTTCGCAGATCGATTACCTCCTCGTTCACTCGTTCGACGCTCAAAAG
GTGATGTTACCGTGTTTTTCAGCAAAAGCCTTGCAGCAAGCCTGCCGGCCCTTC
GTCCTTTCCTGCTGGACGGGCGCCTTGCAGAACAGGGTCTGATCGATCGAGCAA
AACTGGAACCTCTGCTGCACCCCGAACCGATGATTTGGCGCGACTCAGTCGGCG
AGGTAATGCTGGCAGCGTATCTTGAAGCCTGGGTGCGCGCATGGGAAGCCAAGT
TGCGTGTTAGCTAATAA
CGGTAATGGTCGAAGATGATCTGGTTCTGCTGGATGAAGCAGCGGACGCTTATG
TCTGTTTGTTGGATGGCGCCAAAGTGGTTAGCGTCCGGGCTGACGGTGCTCTGA
GCTTCAATCCCCCACATGCAGCAGAAGATATGATCGCGGGTGGCCTCGTCGAAC
CTTCATCAAGTGCCGCGGCGTCAGCAAACCCGCCGGCAAAACTCCCATGTACTC
CGCTGGCGCGCTTATCGCGCCCGCGGCATGTAAAAGTGCGTCCGGCTGAAGCGG
CCTTGTTCCTGATCCAAGCCTGGGGTGTTGCGCGTGCGGTACGTCGTTGGCCAA
TGGCTAGATTATTAGAAGCATTACGTGGAGATCGTGCCGCAGAACCGGCGAAAG
GCCGCCGATCGATGGCGGAGGCGTGCGCTGTTTTTGATGCGCTTCTGGCCTGGA
GCCCTTTTGACGGTGAATGTTTGTTTCGCTCAGTATTACGACGTAGATTTTTAA
TGGCACTGGGCCATTCGCCGGACTTGGTGATAGGCGTGCGTACCTGGCCGTTCC
GCGCACATTGCTGGCTGCAGAGCGGAGTGGATGCCCTGGATGATTGGCCGGAAC
GGCTCTGCGCATATCGCCCGATTCTGGCAGCTTCTGCAAGCCAGGGTAGATAAT
TGGCCGCCGGGGCAGCCGAGCGTAGAAGCTGATGCACTTCACGCAGCCTTTAAC
GGGCAGGGTGGATGGAGCCTGGTTTTGGAACGATTCTGCCTGCGCGTATACGTG
CGTGGCGCGGCAGCCCCTGCAGTTACCCTTACCCCGAAAGGAGGCGTGCTCATT
GGTGAGATGTTTGATCGGGCTGCCACAGAAACGGGCGCCGTTGCCGCTTATGAT
CTGAGCCGCCTGGGAGATGACGACGGTATGGCCGTAGCCCGGCGTGTGGTGGAC
GAAGCGTGGGGGAGATATGTGTTGGTGCTGCCAGTTAAAGAACGCCGTCCAGTG
GTTTTGCGAGAACCACTGGGCGCGCTGGATGCGCTGATCTGGCGCAAAGGCGAT
GTCTGGTGCGTGGGGGCAGACGTACCCCCGGGTCTTGAACCAAAAGATCTGGGT
GTGGAAGAGACTAGACTGACGCACCTGATCGCGGAACCGGATCTGGCATCTGCG
AGCCTGCCCTTAACCGGCGTCGCGGCAGTGATGCCAGGTACTGCGGTCGATGAA
ACCGGCCAGGTGCACCGTCTGTGGACCCCCGCGCGTTTTGCTCGCTCCCCTCGC
ACTGACGCGTGGACTGCAGCCGAACGTATTCCGCTGGTTACCCGTGCGTGCATC
GCGGCGCTGTCTGCGAATCGAAGTGGTATTCTGTGCGAGATTTCGGGCGGCCTG
GATAGCGCTATTGTTGCGACCTCTCTGAAAGCGGAAGGTGCGAAGATTAGTAGC
GGGATCAACTTCCATTGGCCCCAGGCTGAAGCAGATGAGCGCCCGTACGCACGC
GCTGTTGCGAAAAGCGTGCGAACCCGGTTACAGGTGGTAGCGAGTCGTGTAGCG
CCCGTTGACCCGGAAACGTTTGATGAGATCGTGGTCGCGCGACCAAGTTTTAAT
GCCATTGATCCAGTCTATGATACCGTACTGGCCCAACGTCTGATTCAGGGCGGT
GAAGGAGCCCTGTTTACCGGACAAGGTGGTGACGCAGTTTTCTATCAGATGCCA
GCACCACAACTTTCGTTGGATTTGTTGGCTCGTGGCCCCCGCCGCCGCGGTCTT
ATGGGATTATCACGCCGCACCAACCGCAGTGTCTGGTCGTTGCTGCGCATGGGC
TTACGTGCACCCGTACGAGCAACCTTTCCCTACGGTGCGAGAGGTGCCGATCGT
CCTCCGATGCACCCGTGGCTGGAGGACGCGCGTGGTGTTGGGGCCGCGAAACGG
ATTCAGATCGAAGCGCTGGTTGCTAACCAGGCCGTGTTTGAAGCATCTCGTCGC
GGTGCGGCGGCTCATTTGGTGCACCCACTGCTGTCGCAACCGCTTGTGGAGCTG
TGCCTTTCAACCCCAGCGGCCGTGCTGGCGGGTGCCGAACAAGATAGAGCATTC
GTGCGTAGCGCTTTTCGTGCGCAACTGCCACGCCTGGTCTTAGATCGTCAAAGC
AAAGGAGATCTGAGCGTTTTCTTTGCTAAAGGTGTGGCGCGGAGCTTGCCGGGC
TTGCGTCCGCGTCTGCTCGAAGGACGCTTAGCGGCACGTGGCCTGATCGACGTG
GAAGCGTTATCACAAGCGATGCAGCCAGAAGCGATGATTTGGCGTGACGGTTCG
GCCGAAATCCTGTGCCTTGCTGTTCTGGAATCATGGCTCCGCTCTTGGGAGGCT
CGTGGTGCATAATAA
ATTGCGTAAAACAAGGTGGAGTTACGTTTCTGGACGTCCGCGGGGATCGTTACT
TCGGCCTGCCGCCGGTGCTGGAACACGCGTTCGTTGCCATTGCCGAGGCGGATT
TTCTGCTGAAAGAACCAAATTCACTTCTGGAGCCACTCGAAGCACTGGGTGTCT
TAGTGCGAGGCCAAGCCCGCCGTGCCGATCTGACAATTCCGTCTGCAAATCTGT
CATGGGTGGATGAGGTCAGCCCGACCCCACCACGTCTTGACCCTGCGTCACTCG
TCGCAACCGTCACGTCTGTTATTCGAACGCGTCTGAGCCAAAAGAGTAAGTCCT
TGCAGGCTCTCTTGGAAGAGGTCCGTACCCGCCGTCCGGGATCGCCGGCCCATA
ATTGGCAGCTGATGCGTCGTCTGACGGCTGGATTCCGTGCATCGCGTGCTTGGG
CGCCGATAGAACCCATCTGCCTCCTGGACAGCTTGGCGTTACTGGATTTTCTGC
ATCGCCGTGGCCTGTATCCGCATATTGTTTTCGGTGTGATCCGCCAACCGTTTG
CCGCTCATTGTTGGGTGCAAGCTGATGATGTAGTCCTGAATGACCGGCTGGATC
CACGCGACTTGATTCGTCGCCTGCCGAAACTCAAAACCGTCATTGAAACTAGCG
GATTGGTGGTACTGCGCCCCGAAAATGGTGCGGGTCTGCGGGTAGGCGGGAACG
GTGTGGTCCTGGGTAGCGTCTTTCGCACCGGCGGTGATCGCGAAACTGTTGCGG
AATTTTCGGAATCGGAAGCATCCGCGATCGCCACGAGTCGTGGTCAGCAGTTAG
TGACAGAGTTCTGGGGTGGCTACCTGGCTGTTCTTGGAGATGCTTCGCGTTCCG
AAGTGATGGTCCTGCGAGATCCTTCAGGTGCAATGCCGGCTTATTGTTTAGTTC
ATGGCGAAGTCCAGATCATCTGCTCTCGCTTGGAGGTCCTGGAGGACGCAGGAC
TGGGGCAGCAGGCGCTGAACTGGGACGTGGTGGCGCAATTACTGGCCTTCCCAA
ACCTTCGAGGTCGCTCAACGGGTCTTAAAGGCGTGGAAGAATTACTTCCCGGTT
GCCGTCTGACATTTACGGGAGGACTGAAAACCGAAACGCTGACCTGGAACCCGT
GGCTTTTTGCCCGCCCATCTGCGCAAGCGCCTGAACGTGGAGTTGCGGCGACCG
CCGTGCGTCAGGCGGTGGAAGTAAGCGTTCGAAAATGGGCTGATCAGAGTTCAC
CGGTACTTTTGGAATTGTCAGGCGGGCTGGATAGTAGTATCATCGCCTGCTGTC
TGGACGAACCGCGCACCGCGGCCACCTTCGTGAACTTTGTCACACCGACGGCCG
AAGGCGATGAACGAGGATATGCACGTCTGGTTGCCAAGGCAGCAGATAAACAAC
TGATCGAGCAGGACATCCGGGCTGACGAAGTAGATGTTACCCGTCCAAGACCTG
GCCGCCATCCTCGTCCGGCCAGTCAGGCGCTGTTACAGCCGCTGGAACAGGCTT
GCGCTGAACTGGCACCTCAGTTGGGTGCGAGAAGTTTCTTCTCCGGTCTGGGAG
GAGACAACGTGTTTTGTAGCATTGCAACCGCAAGCCCGGCTGCGGATGCACTTT
TGACTAGCGGTCTGGGCCGACAGTTCTGGGCCGCAATCGGGGACCTGTGTGCAC
GTCATAACTGCACCGTATGGGCAGCCTTAAGCGCCACGCTGAAGAAACTGCTCC
GCTCAGATCGTCGTCTGGTGATCAAACCAAACCTGGATTTTCTGTCCTTTCGGG
AGGACGCCATAGACCGTCCGGATCACCCATGGCTTGAAGTGGCCGCCGATCGTC
TGCCGGGGAAACGCGAACATGTCGCAAGCATTCTGTTGGCGCAAGGCTTCCTGG
ATCGTTATGAGCACGCTCAGGTTGCTGCCGTCCGCTTTCCCTTGTTAACGCAAC
CGGTTATGGAGGCTTGTCTGCGCGTGCCGACCTGGATGGCAAACCACCAGGGTC
GCAATCGGGCGGTCGCACGCGATGCCTTCTTTGATCGCTTGCCCCCGAGAGTAC
GTGATCGGCAGACAAAAGGAGGTTTGAACGCGTTTATGGGTGTTGCGTTCGAAC
GCAACCGTCAGGCCTTAGCTCGTCATCTGTTAGACGGGCGCCTGGTACAGCGTG
GCCTGATAGATGCAGTGGCAATAAAATCGGCGCTGGCCTCACCAGTCCTGGAAG
GAGGAGCCATGAACCGCTTACTGTACCTGGCCGATGTCGAATCCTGGGTACGCT
CATGGGAAGATGTGTAATAA
TCTATGCTGTCATGATCGATGATGATGTAGTTTTCCTGGACGTCGCCACCAATG
CATACTTCTGCCTCCCAGCCGTTGGGAGCGTGTTGGCACTCGAAGGTCGTTCGC
TGCGTGTGGCGGCTCGCGAACTGGCAGAAGATCTTATTCAGGCAGGCTTAGCAT
CCGCGGCTGCGGCAATCGAACCCCCACCGAGCACACCAGCCCCAGTTCGCACTG
CGCGTGCGGTATTGGAAGCTCTGCCGGCGCGTGAAAGACCACGTCCACGTCTTG
CCCACTGGCGTCAGGCGATTATGGCTGGCTTGGCGTCCCGTGCCGCTGAACGTC
GACCATTCGCGCAGAGACTGCCGCCGCCTTCAACGGGGGTTTCACCTCCGGCAT
CAGAAGGCCTGCTTGCCGATCTGGATGCGTTCCGTCGACTTCAGCCATGGTTGC
CGTTCGACGGTGCTTGTCTGTTCCGTAGCCAAATGCTGCGCGATTATCTCCTTG
CGCTGGGTCACCGCGTTGACTGGATTTTCGGTGTACGTACGTGGCCGTTTGGTG
CCCACTGTTGGTTGCAGGCCGGCGACCTGGTGCTGGATGATGAGGCCGAACGTC
AGCAGCGTTTGATGAGATGGTAGAAGCACTGATCGATGCTGGATGGACCTTGGC
GTTGCGTGCGTTCAGACTCGCCGTTCTCACCGATGGTCAGGCTCCAGCCGTGTC
GCCGCTGATGGGCAGAGGCGGCGTAGCAGGCGTTCTCATCGGCGAAGCGTTTGA
TCGTCGCGCCACATTAGGTGGCGCGGTCGCACGTGCCGCGCTGGATGGTTTGGC
TGACATCGATCCGCTGGAAGCAGGTCGCCATCTGATTGAAACCGCGTGGGGCGG
CTACGTGGGTATGTGGATTGGTCGGGCCGAAGCTGGTCCGACACTGCTGCGCGA
TCCTAGTGGCGCGCTCGAAGCCTTAGCGTGGCGCCGTGACGGTGTAACCGTTAT
GTCAGCGCGCCCGTTGACGGGGCGCGCAGGCCCAGCTGATTTAGCAATCGATTG
GCCACGTATCGTGCAGATTCTGGCCGATCCCATTTCCGCGGCTCTCGGCCCGCC
CCCTCTGACTGGCTTAGCGACCATAGACCCGGGCGCGGCGGTTCATGGCGCGGA
TGGCCAAGAACGCTCAGTGCTGTGGACCCCAGCTGCAGTTGTCCGTGGTGCTCG
TCACCGTCCTTGGCCAAGCCGTCAGGATCTGCGTCGCACCATCGATGCGACTGT
CGCGGCACTGGCCTCGGATGCGGGCCCGATTGTCTGCGAAATTTCAGGAGGTCT
GGACTCGGCCATAGTTGCGACTAGCCTTGCGGCGTCCGGTCTGGGTCCGCAGCT
GACAGTGAATTTTTACGGTGACCAGCCTGAAGCTGATGAACGCGGATACGCTCA
AGCCGTCGCCGAACGTATCGGTGCGCCTCTGCGGACCCTTCGTCGAGAGCCGTT
CGCGTTCGATGAAACCGTGCTGGCAGCCGCTGGACAGGCCGCACGTCCGAATTT
TAACGCCCTCGATCCTGGATACGATGCCGGGCTCGTGGGTGCCCTGGAAGCTAT
CGATGCTCGTGCATTATTTACGGGCCATGGCGGTGATACCGTGTTTTATCAAGT
GGCGGCCAGTGCCTTGGCCGCAGACTTACTGGGCGGCGCACCATGTGAAGGTAG
CCGCCGTGCACGTTTAGAGGAAGTAGCTCGGCGGACCCGACGCTCGATTTGGAG
TCTTGCATGGGAAGCGTTTTCTGGTCGACCCAGCACTGTAAGCATTGAAGGTCA
GTTGCTTCGACAGGAAGCAGAGAGAATTCGGCGCGTCGGCCTGACCCATCCGTG
GGTTGGAGGCCTGTCGTCTGTGACCCCTGCGAAACGCCAGCAAATCCGCGCGCT
GGTCAGTAACCTGAACGCGCATGGCGCCACTGGTCGCGCCGAACGCGCTAGAAT
CGTGCACCCGCTTTTAGCTCAGCCGGTGGTTGAAGCCTGCCTGGCGATTCCTGC
CCCTATCCTCAGTGCGGGCGAAGGAGAACGCTCATTTGCGAGAGAAGCCTTTGC
AGACCGTTTGCCACCGAGCATTGTGGGCCGCCGAAGCAAAGGGGAAATTAGTGT
GTTTCTTAACAGATCTTTAGCAGCCAGCGCCCCCTTTCTGCGTGGCTTTTTACT
TGAAGGACGGCTGGCGGCTCGCGGGCTGATTGATCGTGACGAACTTGCAGCCGC
GCTGGAACCGGAAGCAATCGTCTGGAAGGATGCGTCACGCGACCTGCTTACTGC
GGCGGCCCTGGAGGCGTGGGTCAGACATTGGGAAGCACGTATTGGCGAGGGGGA
AGCAGCGGAAGGTGAGCGTGCTGCCGGTCGTGGTACCGCAGCGACGGGACCGCG
TACAAGCGCGCGGAAGGCGAACACCCGTTAATAA
ACCATTATAAAGCCTTTGGGTTTAGAATTGAAAGCGATTTCGTGCTCCCGGAAC
TTCCGCCCGCAGGCGAACGCGAACCGCTCGATAATATTACGGTTCGTCGTACCG
ACCTGCAGCCGCTCTGGAATTCTAGTATCCATTTTTACGGAAACTTTGCCATTC
TGGATCACGGACGCACGGTTATGTTTCGAGTTCCGGGTGCTGCTATCTATGCGG
TACAGGATGCTAGCAGCATATTAGTGTCCCCATTCGATCAGGCAGAAGAAAACT
GGGTACGTCTTTTTATTCTGGGTACCTGTATTGGGATCATCCTGCTGCAGCGTA
AGATTATGCCGCTGCACGGTAGCGCCGTTGCCATTGATGGCAAAGCCTACGCGA
TTATCGGCGAATCTGGTGCCGGCAAAAGCACTCTTGCACTGCATCTTGTCAGTA
AGGGTTATCCATTGCTTTCGGATGATGTGATTCCGGTCGTTATGACCCAGGGCT
CCCCCTGGGTGGTGCCGTCGTACCCGCAACAAAAACTTTGGGTGGACACTCTGA
AGCACATGGGAATGGATAATGCAAACTATACGCCGCTGTACGAACGTAAAACGA
AGTTCGCGGTGCCCGTGGGCAGTAATTTCCACGAAGAACCGCTGCCGTTAGCTA
GCATTTTCGAGCTTGTCCCGTGGGATGCGGCAACGCACATTGCCCCGATCCAAG
GGATGGAACGCTTTCGTGTCCTGTTCCACCACACTTATCGGAACTTTCTGGTTC
AGCCGCTGGGTCTTATGGAATGGCATTTTAAAACTCTGAGCTCGTTCGTTCACC
AAATTGGAATGTATCGTCTGCATAGACCTATGGTCGGATTCAGTACCTTAGATT
TAACGTCGCACATTCTGAATATAACGCGTCAGGGAGAGAACGATCAATAATAA
TCGCGCGTTCGGCCTGCGCATAGACTCAGATATTCCGCTGCCAGAATTAGGGGA
CGGTACGCGCCCTGATGGTGACGCGGATCTGACGGTCGTCCGGTGTGGGGAAGC
GGAGCCGGAATGGGCTGAAGGTGGTGGCGGGGGTCGTCTGTATGCCGCTGAAGG
CATTGTATCTTTTCGCGTGCCGCAGACGGCAGCGTTCCGTATTACTAATGGAAA
TCGCATCGAGGTGCATGCCTACTCGGGGGCTGATGAGGATCGAATACGCCTGTA
CGTGTTAGGGACCTGTATGGGAGCGCTGTTACTGCAACGTAGAATCTTACCGCT
TCATGGTTCGGTCGTCGCCCGTGATGGTCGTGCGTATGCCATAGTTGGCGAAAG
CGGAGCGGGCAAATCCACGATGAGTGCAGCACTTCTCGAACGTGGATTCCGCCT
CGTTACGGATGACGTGGCCGCCATCGTGTTCGATGAGCGTGGGACCCCACTGGT
TATGCCGGCTTATCCACAGCAAAAACTGTGGCAGGATTCCCTGGACCGTCTGCA
AATTGCGGGCTCGGGCCTTCGTCCGCTGTTCGAACGCGAAACGAAATACGCTGT
ACCCGCGGATGGGGCATTCTGGCCCGAACCGGTTCCATTGGTGCACATTTACGA
ACTGGTTCATAGCGATGGTCAAACGCCTGAACTGCAGCCGATTGCCAAATTAGA
GCGTTGCTATACCTTGTATCGCCACACATTTCGTAGAAGCCTGATCGTCCCCAG
CGGCTTAAGCGCCTGGCATTTTGAAACGGCAGTGAAACTTGCGGAGAAAACGGG
GATGTACCGTCTTATGCGCCCGGCCAAAGTTTTCGCGGCTCGCGAATCTGCTCG
GCTGATTGAAACTCACGCCGATGGTGAAGTGTCACGTTAATAA
CACCGTCCTGAGCCTGGCCGAACGGACAGGTACCGATCCAGATCTGCTGGGCCG
TGTGTTGCGCTTCCTCGCTTGTCGTGGTGTTTTCGCCGAGCCTCGCCCAGGTAC
TTATGCCTTGACCCCTCTGAGCTTAACTTTACTGGAAGGCCATCCGTCCGGTTT
AAGAGAATGGTTGGATGCGTCGGGTGCGGGAGCGCGCATGGACGCGGCAGTTGG
AGATCTGCTTGGCGCCCTCCGCTCGGGTGAACCGAGCTATCCACGTCTGCATGG
TCGTCCGTTTTATGAAGATCTGGCGCTGCACAGCCGAGGCCCTGCTTTTGATGG
ACTGCGTCATACGCACGCCGAATCGTATGTTGCCGACCTGCTGGCAGCCTACCC
GTGGGAACGCGTTCGTCGCGTGGTTGATGTAGGCGGTGGGACCGGCGTATTGGT
CGAGGCGCTTATGAGAACTCATGCGACCCTCCGTACAGTACTGGTCGATCTTCC
AGGCGCGGTGGCTACCGCTACCGCTCGAATTGCGGCTGCGGGTTTTGGCAATAG
ATATACACCGGTCACGGGTTCCTTCTTTGATCCGCTGCCTGCGGGGGCGGATGT
TTACACCCTGGTTAACGTGGTTCACAACTGGAACGATGAGCGTGCCTCAGCTCT
GCTGCGTCGGTGTGCGGATGCGGGTCGCCGCGACAGTACGTTTGTTATCGTGGA
ACGCTTAGCGGACGATGCAGACCCTCGTGCCATCACCGCCATGGACCTCCGTAT
GTTCCTTTTTCTGGGCGGTAAAGAGCGCACGGCCGCACAGATTCGCGAAGTAGC
TAGTGCGGCTGGCATGGCCCACCAAAGCACCATTAAAACACCGTCTGGCCTCCA
CTTACTTGTTTTCCGTAAGAAACGTTTCGCTGCTCGCGGTCACGGTCGTCGCAT
GGTGACCTAATAA
ACAAGTGGTTTGATATTAACTTCCTGGAAATGTATACACGCAGCTGCCTGAAAA
CTTTTGGCTACTTCGACGAAATTCTGATCGTGAAGAAACGCATCGAGGTCCTGA
AGAACGTGCTTGAAAAACAGTACTTGTCTACCAATGATTATGCTGAGGAGTTTT
TCGAGCTGAATACCACCTTGGAGAGCATAAAAGAATACATCAAACTGAATCTGG
TCATCGAGAAAGAACCGATCTCAATTTGCATTATGGTCAAAAACGAAGAACGTT
GCATCAAGCGCTGCATTGATAGCGTTGAAATCCTCGCCGAGGAGATAATCATTA
TCGATACCGGCTCTACGGATAATACCATTAACATTATTGAGGAATGCGCAAACG
ACAAAATTAAAGTGTTCTCAAAAGAATGGCGTAACGATTTTTCCGAAATTCGGA
ACTATGCCATCGAGAAAGCGAGTAGCGAATGGCTGGTGTTTATAGATGCCGATG
AATATCTGGACGAAGCCTCGGTGCTCAACCTGCTCAGTACGCTCAACATCTTTA
ACAATCATAAGCTCAAAGACTCTATTGTCCTGTGCCCCATGATCAACGAAGCCA
ATAACACCATCCATTTCCGTACCGGGAAATTTTTCAGAAAAGACTCCGGGATTA
AATTCTTTGGTACCTGCCATGAGGAGCCCCGCATTAAAGGCATGCCGAATTCTA
CCCTGCTGATTCCGATCAAGGTTGATTATCTGCATGACGGCTACCTGGCAAAAG
TACAATCAAATAAAGACAAGAAAACCCGTAACATCGAACTGTTAGAAGGTATGG
TGGAACTGGAACCGGATAATCCTCGTTGGGCGTATATGTTTGTGCGCGACGGAT
TTGCAATCCTCGATAACGAATACATTGAGAAAACTTGTTTGCGGTTTTTACTGC
TGGACAAAAACGTACGCATCTGCGTCAACAACCTGCAAGACCATAAATTCACTT
TGTCACTCCTGACGATCCTGGGCCGCCTCTATCTGCGCGAGTGCGAATTCGAGA
AAAGCAATCTGATAATTCGCATTCTTGACGAACTCATCCCTAATAGTCTGGATG
GTAAATTTCTGGCATTCATGGAGCGATTCAGCAAACTGAAAATTGAGATTAATA
CGCTGTTAACGGAGGTCATCGAATATCGTCGTAACCACGAAGTAGATGAAACCA
GTTTAATCAACACACAAGGCTACCATATCGACTATGTTCTGTCGATTTTGCTGT
TCGAAACGGGTAATTACGCGCAAAGTAAGAAATACTTCGATTTCCTGCAGGAGA
ACCATTTTCTGGAAGAACTGTTTCAAGACAGCTCTTATTCTATCATACTGAAAA
TGCTCGAGTCAGTAGAAGATTAATAA
GATGAAAGATAACTATGCGGACTCTAATCTGTTCAAGGATTTGAATCTGATCCA
CAATATCTCCAACGACATCCAAATTGGAATTAATTGCGATTTCTCTGAAATGCT
GGGAGAACTGGTAGGTAATTACGATTCCCTGAACTATCCGTCAATCACCTGTGG
TATTCTGACGTATAATGAAGAACGCTGCATTAAACGTTGTCTGGAAAGTGTGGT
GAACGAATTCGATGAGATTATTGTCTTGGATAGTGTATCCGAGGACAATACCGT
GAAAATTATCAAGGAGAATTTCAACGATGTCAAAGTCTACGTCGAGCCATGGAA
GAACGATTTTTCATTTCACCGCAACAAGATCATTAATCTCGCAACGTGCGACTG
GATCTACTTTATCGACGCGGATAATTATTATGATTCGAAGAACAAGGGTAAAGC
CATGCGCATCGCTAAGGTTATGGATTTCTTGAAAATCGAAGGCGTTGTGAGCCC
AACGGTCATTGAGCATGACAATAGCATGAGCCGTGATACCCGTAAGATGTTTCG
TCTGAAAGATAACATTCTGTTTAGCGGTAAAGTTCATGAAGAACCGGTGTATGC
CAATGGTGAGATCCCCCGGAACATCATAGTAGACATCAACGTGTTTCACGACGG
CTATAACCCAAAGATTATCAACATGATGGAAAAGAACGAGCGCAATATCACCCT
GACTAAAGAGATGATGAAGATCGAACCGAACAATCCGAAATGGCTGTACTTCTA
TAGCCGCGAACTCTATCAGACGCAACGTGACATTGCCCTTGTGCAAAGTGTACT
GTTCAAGGCACTGGAACTGTATGAAAACAGTTCATATACGCGTTATTATGTTGA
CACCATTGCCTTACTGTGCCGAGTGCTGTTCGAATCTAAAAACTACCAGAAACT
TACGGAATGTCTGAACATCCTGGAGAACAATACGCTTAACTGTTCCGATATCGA
TTACTATAATTCAGCGCTGCTGTTCTACAACCTGTTACTGCGCATCAAGAAAAT
TAGCTCCACCCTGAAGGAGAACATTGATATGTACGAACGTGACTATCATAGCTT
TATCAACCCCTCGCATGATCACATTAAGATTCTGATATTAAATATGCTCCTGCT
GCTCGGCGATTACCAGGATGCCTTTAAGGTTTACAAGGAGATCAAGTCCATTGA
GATTAAAGATGAGTTTCTGGTGAACGTGAACAAATTCAAAGACAATCTTCTGAG
CTTCATTGACTCCATTAACAAAATTTAATAA
GACCTTCTGCGCCAAGCATTACACGCAACTGGTACAGGTGCTCGTTGGGCTGTA
GAGGCGGACGAGATGTGGTGCCGTGTCGCCCCGGTGCCTGGAACTCGCCGCGAG
CAAGGATGGAAGCTTCATGTAAGCGCGACGACCGCGAGTGCGCCCGAAGTCTTA
ACTCGTGCATTAGGCGTACTTCTGCGTGAAAAGTCCGGGTTCAAATTTGCCCGC
TCACTTGAACAAGTCTCGGCCTTGAATAGTCGTGCTACGCCCCGTGGTAGTTCG
GGTAAATTTATCACAGTATACCCCCGCTCAGACGCCGAAGCCGTCGCACTGGCT
CGCGACCTGCATGCGGCAACGGCCGGCTTGGCTGGGCCCCGTATTCTTTCCGAT
CAACCATACGCCGCGCACAGCCTGGTGCATTATCGTTATGGGGCTTTCGTGGGA
CGTCGTCGCCTTTCAGATGACGGGCTTTTAGTTTGGTTTATTGAGGACCCAGAT
GGCAATCCCGTGGAGGATAAACGCACCGGACGTTATGCGCCGCCTCCCTGGGCT
GTATGTCCGTTTCCTGCGAGCGTCCCCGTTGCGCCCCATGACGGCGAAGCTACG
AGTCGTCCTGTTGTCTTAGGTGGTCGCTTCGCGGTTCGTGAAGCCATCCGTCAA
ACGAATAAAGGGGGCGTCTATCGCGGGTCGGACACACGCACTGGCACCGGCGTG
GTTATCAAAGAGGCGCGCCCACATGTTGAAGGAGACGCCAGTGGGGGCGATGTT
CGTGACTGGCTTCGCGCAGAGGCGCGTACGCTTGAAAAATTAAAAGGTACCGGC
TTGGCACCAGAAGCGGTGGCGTTGTTTGAGCACGCTGGCCACTTGTTCTTAGCC
CAAGACGAGGTCCCGGGGGTTACGTTACGCACCTGGGTAGCGGAACACTTCCGT
GACGTTGGAGGAGAGCGCTATCGTGCCGACGCCCTGGCTCAGGTGGCTCGTTTA
GTTGATTTAGTCGCGGCTGCTCATGCACGTGGCTTGGTCCTGCGCGATTTTACA
CCAGGGAACGTGATGGTCCGTCCAGACGGCGAATTGCGCCTTATTGATTTAGAG
CTGGCGGTTCTTGAGGATGAGGCCGCATTGCCTACCCACGTCGGTACCCCGGGG
TTTTCGGCACCCGAACGCCTTGCAGACGCTCCAGTGCGTCCTACTGCTGACTAC
TATTCTCTGGGAGCCACAGCTTGTTTTGTCTTGGCCGGTAAAGTCCCTAATTTA
CTTCCTGAAGAACCCGTGGGTCGCCCATCGGAGGAGCGTCTTGCTGCCTGGTTG
ACTGCATGTACACGTCCGCTGCGCCTGCCAGATGGAGTCGTTGACATGATCTTG
GGGTTAATGCGCGATGATCCTGCAGAGCGCTGGGACCCATCCCGCGCGCGTGAA
GCACTGCGCAAAGCTGACCCGACAGCACGCCCCGGGGATGCTGATCGCACTGCA
GTACGTCGTACGGGTTCGTCGGCAGTGGCCGGGCCAGTTCCTGACTCACGTACA
GCAGATGGTCGTACAGCGGACGGCCGTTCCGCGGATGAAGTTGTGGCAGGTCTT
GTCGATCACTTAGTCGATAGTATGACCCCGGCAGATGATCGTCTGTGGCCGGTA
AGCACTCTTACGGGAGAATCGGATCCATGTACAGTCCAGCAAGGCGCTGCTGGG
GTGCTTGCGGTGTTGACCCGCTACTTCGAATTGACGGGCGATCCGCGCTTACCA
GGCTTATTGTCGACAGCCGGACGTTGGATCGCAGACCGCACGGATGTTCGTTCA
CCTCGTCCGGGATTACATTTCGGGGGACGCGGAACAGCCTGGGCCTTATACGAC
GCGGGGCGTGCAGTCGACGATCGTCGCTTGGTGGAACATGCTCTGGACTTAGCA
TTAGCCCCGCCCCAAGCGACTCCTCATCACGATGTCACGCATGGGACTGCGGGC
TCAGGCTTAGCCGCCTTGCACCTGTGGCAGCGTACTGGAGATACTCGTTTCGCG
GATTTAGCAGTAGAGGCAGCTGATCGCTTAACAGCTGCAGCTCGTCGCGAGCCT
TCGGGTGTTGGATGGGCAGTACCTGCAGAGGCCGACTCCCCAGAAGGAGGCAAG
CGTTACCTGGGCTTCGCTCATGGCGCAGCTGGGATTGGGTGCTTCTTATTGGCT
GCGGCGGAACTTAGTCGTCAACCCGATCATCGTGCAACTGCTTTGGAAGTTGGC
GAAGGCCTGGTTGCTGATGCTGTTCGCATCGGAGAGGCGGCACAGTGGCCTGCG
CAATCCGGGGACTTGCCGACAGCGCCTTACTGGTGCCATGGGGCGGCAGGTATC
GGGACATTTCTTGTACGCTTATGGCAGGCGACCGGGGACGATCGCTTCGGTGAT
CTGGCCCGCGGGAGTGCTCACGCTGTGGCCGAACGTGCTAGTCGCGCCCCATTG
GCGCAATGTCACGGTTTGGCTGGAAACGGAGATTTCTTGTTGGATTTGGCAGAC
GCGACAGGCGATCCTGTGCATCGCGACACCGCGGAAGAGTTAGCAGGGTTGATC
TTGGCCGAAGGAACCCGTCGTCAGGGACATGTCGTTTTCCCTAATGAGTATGGG
GAAGTATCATCTTCATGGTCCGACGGTAGTGCGGGGATTCTTGCGTTCCTTCTG
CGTACGCGTCATACGGGCCCTCGCCATTGGATGGTAGAACAACGTGGGTAATAA
CTGCCTCGTGCGGGGAGTTCATTACTGGCTGCGTTACTGCGTCAAAATCCGCAG
CTGCATGCCGATGTTACATCTCCGGTGGCGCGCCTTTACGCGGCCATGCTGATG
GGTATGAGTGAAGAACACCCGAGCAACGTGCAGATTGACGATGCCCAACGTGTC
CGTCTGTTACGTGCAGTATTTGATGCGTATTATCAGAACCGTCAGGAACTGGGG
ACAGTGTTCGATACTAACCGCGCATGGTGCTCTCGCCTCACGGGCCTGGCGCGT
CTGTTTCCGCGTAGTCGCATGATCTGCTGTGTACGCGATGTGGGCTGGATTGTT
GATTCTTTTGAACGCCTGGCGCAGTCGCAGCCGTTACGCCTTTCGGCCCTGTTC
GGTTACGACCCCGAGGATTCGGTTAGCATGCACGCTGACTTACTCACTGCGCCT
CGCGGGGTAGTGGGCTACGCCCTGGATGGTTTACGTCAAGCGTTTTATGGAGAT
CACGCGGATCGTCTGCTGTTGTTACGTTATGATACGCTGGCACAGCGTCCTGCA
CAAGCCATGGAACAGGTATATGCATTCCTGCAGCTCCCTGCCTTTGCACATGAT
TATGCCGGTGTTCAGGCCGAAGCGGAACGCTTTGATGCCGCCCTGCAAATGCCT
GGTTTGCACCGCGTGCGTCGTGGTGTTCACTATGTTCCGCGACGTTCGGTTTTA
CCGCCTGCCCTGTTTGACCAGCTGCAGGAACTTGCATTCTGGGAAAGTGCACCC
AGCCATGGAGCGCTGCTCGTGTAATAA
TCAGTAACAAAGACCTGTCGCAACTCCTGTGTTCCTTCATTGATTCAAAGGAAA
CTTTCAGTTTTGCCGAGAGCGCTATACTGCATTATGTAGTATTCGGCGGTGAGA
ACCTGGACGTAGCTACCTGGCTGGGCGCCGGAATTGAAATTCTGATCCTGAGCA
GCGATATCATGGACGACCTGGAGGACGAGGATAACCATCATGCGTTGTGGATGA
AAATTAACCGCAGCGAGAGCTTGAATGCGGCCCTGTCCTTATACACCGTCGGCT
TAACGAGCATCTATTCCCTGAACACAAATCCGTTGATATTTAAGTATGTGCTGC
GCTACGTCAATGAGGCCATGCAGGGTCAGCATGATGATATAACCAATAAAAGCA
AAACCGAAGATGAATCGCTTGAAGTGATTCGCCTTAAATGCGGCAGCCTGATCG
CCCTGGCAAATGTCGCGGGCGTGCTGTTAGCCACGGGCGAGTACAATGAAACAG
TTGAACGTTACTCTTATTACAAAGGCATCGTGGCGCAAATTTCCGGCGACTATC
ACGTGCTGCTGTCAGGAAACCGGAGCGATATCGAGAAAAACAAACAGACACTGA
TTTACCTGTATCTGAAACGCCTGTTTAACAACGCGAGCGAGGAATTGCTGTATC
TGTTCTCCCATAAAGATTTGTACTATAAAGCCCTGCTCGACCGTGAAAAGTTTG
AAGAAAAACTGATCCAGGCCGGGGTGACGCAGTACATCAGCGTTCTGCTCGAAA
TATATAAGCAGAAGTGCTTCTCCACCATAGAACAGCTGAACTTAGATAAAGAAA
AGAAAGAGCTGATCAAGGAGAGCCTGCTGTCATATAAGAAAGGCGACACCCGTT
GCAAGACCTAATAA
CTCCATAAGAGTAAAAACTTGATGTATATGAAAGCCCACGAAAACATCTTCGAA
ATCGAGGCGCTGTACCCGCTGGAATTGTTCGAGCGTTTTATGCAGTCCCAAACC
GATTGCTCCATCGATTGTGCCTGTAAAATTGATGGTGACGAATTGTATCCCGCC
CGTTTTAGTCTGGCCCTGTATAACAACCAGTATGCCGAAAAGCAAATTCGCGAA
ACCATCGACTTCTTCCATCAGGTAGAGGGTCGGACCGAGGTGAAACTGAACTAT
CAGCAACTGCAGCACTTCCTGGGTGCTGACTTCGATTTTAGCAAAGTGATTCGA
AACCTGGTGGGTGTGGATGCACGCCGCGAACTGGCTGATTCCCGGGTTAAACTG
TATATTTGGATGAACGATTACCCAGAGAAAATGGCGACCGCCATGGCATGGTGC
GATGATAAGAAGGAATTGTCGACGTTGATAGTAAATCAGGAGTTTCTGGTCGGG
TTCGATTTTTATTTCGATGGTCGCACGGCAATAGAATTATACATTAGTCTGTCA
TCCGAAGAATTTCAGCAGACACAAGTTTGGGAACGCCTCGCAAAGGTAGTGTGC
GCCCCAGCGCTGCGCCTTGTTAATGATTGCCAGGCGATCCAGATTGGCGTGAGC
CGTGCCAATGATAGTAAGATCATGTATTACCATACCCTTAATCCGAACTCGTTT
ATCGACAATCTGGGCAATGAAATGGCAAGCAGAGTTCACGCGTATTACCGACAT
CAACCGGTTCGCTCTCTGGTAGTATGCATACCAGAACAGGAGTTGACCGCCCGG
TCCATACAGCGCTTAAACATGTATTACTGTATGAACTAATAA
CCTGGATCTGACCGTGGATTATATTATTAATCGCTATAATCATACCGCTAAATT
TTTTCGTCTGAATACCGATCGTTTTTTTGATTATGATATTAATATTACCAATAG
CGGTACCAGCATTCGTAATCGTAAATCTAATCTGATTATTAATATTCAGGAAAT
TCATAGCCTGTATTATCGCAAAATTACCCTGCCGAATCTGGATGGCTATGAAAG
TAAATATTGGACCCTGATGCAGCGCGAAATGATGAGTATTGTTGAAGGCATTGC
AGAAACCGCTGGCAATTTTGCACTGACCCGTCCGTCTGTGCTGCGCAAAGCTGA
TAATAAAATTGTGCAGATGAAACTGGCAGAAGAAATTGGTTTTATTCTGCCGCA
GAGTCTGATTACCAATTCAAATCAGGCGGCAGCCTCATTTTGCAATAAAAATAA
TACCAGCATTGTGAAACCGCTGAGTACCGGCCGCATTCTGGGTAAAAATAAAAT
TGGCATTATTCAGACCAATCTGGTTGAAACCCATGAAAATATTCAGGGCCTGGA
ACTGTCTCCGGCTTATTTTCAGGATTATATTCCGAAAGATACCGAAATTCGTCT
GACCATTGTTGGTAATAAACTGTTTGGCGCCAATATTAAATCAACCAATCAGGT
TGATTGGCGCAAAAATGATGCACTGCTGGAATATAAACCGGCCAATATTCCGGA
TAAAATTGCCAAAATGTGTCTGGAAATGATGGAAAAACTGGAAATTAATTTTGC
GGCGTTTGATTTTATTATTCGTAATGGTGATTATATTTTTCTGGAACTGAATGC
CAATGGTCAGTGGCTGTGGCTGGAAGATATTCTGAAATTTGATATTTCAAATAC
CATTATTAATTATCTGCTGGGTGAACCGATTTAATAATAA
TGATTCATTTCCATCCGTACAAACTGTTCGAGGTGGATTCAAAAACCTTCTTCT
ATAACGTAGTCACCAACGCGATTTTTGAAATTGATAGCCTGATAATCGACATTC
TTCACTCAAAAGGTAAAAATGAGGAGCACGTTGTGAAAGATTTGGCTGAACGCT
ATGAGCTGTCTCAGGTTCGCGAAGCGATCCAGAACATGAAAGAGGCATACATTA
TAGCAACCGATGCTAACATCTCCGACGTAGAGAAGATGGGTATCTTAGATAACT
CGCAGCGCGTTTTTAAACTGTCTAGCCTGACGCTCTTTATGGTGCAGGAATGCA
ACCTGCGGTGTACGTATTGTTACGGCGAAGAAGGAGAATACAACCAGAAAGGTA
AAATGACGTCCGAAATCGCCCGGAGCGCAGTGGATTTTCTGATTCAACAGAGTG
GTGAAATCGAACAGTTGAACATCACATTCTTTGGAGGCGAACCGCTGCTCAACT
TTCCATTAATACAAGAAACCGTGCAGTATGTGCACGAACAGAGCGAGATCCATA
ACAAGAAATTTAGCTTTTCCATCACCACCAATGGCACGCTCATTACCCCCAAAA
TCAAAAACTTCTTCTATAAACACCACTTTGCAGTCCAGACTTCTATCGATGGTG
ATGAAAAGACGCACAATTTCAATCGCTTCTTCAAAGGAGGCCAGGGCTCTTATG
ATCTGCTGTTAAAGCGGACGGAAGAAATGCGCAATGACCGTAAAATTGGTGCAC
GTGGAACCGTGACCCCTGCCGAGCTGGACCTCTCAAAATCATTTGACCACTTAG
TTAAACTCGGCTTTCGCAAAATCTACTTATCACCCGCTTTATATAGTCTCTCTG
ACGATCACTACGACACCCTGAGCAAAGAGATGGTCAAACTTGTTGAACAATTCC
GTGAGCTGCTGGAGCGTGAAGATTACGTCACCGCGAAGAAAATGTCTAATGTTC
TGGGTATGTTATCGAAGATTCACTCCGGTGGCCCGCGCATTCATTTTTGCGGTG
CCGGCACTAATGCTGCCGCTGTCGATGTCCGCGGCAACCTTTTCCCGTGTCATC
GTTTCGTGGGTGAAGATGAATGTTCAATCGGTAACCTGTTCGACGAGGACCCGC
TGTCAAAACAGTACAACTTTATAGAGAATTCTACAGTACGCAACCGTACTACGT
GTTCGAAATGCTGGGCGAAGAATCTGTGCGGCGGTGGTTGTCACCAAGAAAATT
TCGCCGAGAATGGTAATGTGAACCAGCCAGTGGGCAAATTATGCAAAGTGACCA
AAAACTTCATCAACGCGACCATCAATCTGTACTTGCAACTTACTCAAGAACAAC
GCAGCATTCTGTTCGGCTAATAA
AGATCACGCTGAAATGCAATCTGGCATGTTCGCACTGTGGAAGTCGTGCCGGGC
ACACGCGAGCAAAAGAACTGTCCACACAGGAAGCGCTGGATCTGGTCCGTCAGA
TGGCTGATGTCGGCATTATCGAAGTTACTCTGATTGGGGGTGAAGCGTTCCTGC
GTCCAGACTGGCTGCAGATTGCCGAGGCGATAACGAAAGCCGGGATGCTGTGCA
GCATGACTACGGGCGGTTATGGCATATCGCTGGAAACCGCCCGCAAAATGAAAG
CGGCAGGAATCGCGAGCGTGAGCGTTAGCATCGATGGCTTGGAGGAAACCCATG
ATCGCTTACGCGGTCGCAAAGGCTCTTGGCAGGCTGCGTTTAAAACAATGAGCC
ATTTGAGAGAAGTGGGCATCTTCTTTGGCTGTAACACCCAGATTAACCGTCTGT
CGGCCCCTGAATTTCCGCTGATATATGAACGCATCCGTGACGCCGGGGCACGTG
CCTGGCAGATCCAGCTTACGGTGCCGATGGGCCGCGCTGCCGATAACGCAAATA
TCCTTCTGCAACCGTACGAACTGCTTGATCTGTATCCGATGATTGCTCGAGTGG
CCCGCCGGGCCCGTCAAGAGGGCGTGCAAATCCAGCCAGGTAATAATATTGGGT
ATTACGGCCCTTACGAACGTCTTTTACGTGGCCGGGGGAGCGATAGTGAGTGGG
cattttggcagggctgtgccgcgggcttaagtaccctgggtattgaagcggatg
GTGCTATAAAAGGTTGTCCCTCACTGCCAACGAGCGCGTATACCGGCGGTAACA
TTCGCGAACATAGTCTGCGAGAAATAGTGGAAGAATCGGAACAGCTGCGTTTTA
ACCTCGGTGCAGGGACGAGCCAAGGGACCGCCCACTTGTGGGGCTTTTGCCAGA
CGTGTGAATTTAGTGAATTGTGCAGAGGTGGTTGTACGTGGACAGCTCACGTGT
TCTTTAACCGCCGTGGGAATAACCCGTATTGTCATCATCGGGCGCTTTTCCAAG
CGGAGCAGGGTATCAGAGAACGTGTCGTGCCAAAGGTCGAAGCTCAGGGCCTGC
CGTTTGACAACGGTGAATTTGAACTTATCGAAGAACCTATTGACGCGCCTCTGC
CCGAAAATGATCCACTGCACTTTACCAGCGACTTAGTGCAGTGGTCAGCGAGTT
TGAACCGGAAAGCCTGCTTCTGCCGCGCCAGGCTTGGCAGTCGCAGATCGCCTA
TCTTAAAGCGATTCTGAAAGCCAAACAGGCGCTTGACCGGATCGAAAAACGTTA
TCTGCGGTAATAA
ACATTTCCATGTAGAGGTCATTGAACCAAAGCAAGTCTACTTGTTGGGTGAACA
AGCTAATCATGCATTGACAGGCCAATTATACTGCCAAATTTTGCCATTGTTAAA
CGGACAATACACATTGGAACAAATCGTTGAAAAACTAGACGGAGAAGTACCACC
TGAATACATTGATTATGTGCTGGAGAGACTAGCTGAGAAGGGCTATCTGACTGA
AGCAGCACCTGAATTATCTAGTGAAGTGGCCGCTTTCTGGTCTGAGCTGGGGAT
TGCACCTCCTGTCGCGGCCGAAGCATTACGTCAACCTGTGACTTTAACACCTGT
TGGAAACATCAGCGAAGTAACAGTAGCAGCCTTAACCACAGCCCTACGTGATAT
CGGTATTTCCGTTCAAACACCTACAGAAGCTGGATCGCCAACTGCATTGAACGT
TGTACTTACCGATGATTATCTCCAACCAGAACTCGCTAAGATCAATAAGCAAGC
CTTAGAAAGTCAACAAACTTGGCTACTTGTCAAACCAGTTGGCTCCGTGTTATG
GTTGGGTCCGGTATTCGTGCCAGGAAAAACAGGTTGCTGGGATTGTTTGGCTCA
CAGATTAAGGGGGAATAGAGAGGTAGAGGCCTCTGTATTGAGACAAAAACAAGC
TCAACAACAACGTAATGGACAAAGCGGGTCTGTAATAGGATGCCTTCCCACGGC
TAGAGCGACACTGCCCTCAACACTCCAAACTGGGCTGCAGTTCGCTGCTACCGA
AATTGCTAAATGGATAGTTAAGTATCATGTTAATGCCACAGCGCCTGGCACCGT
ATTCTTCCCTACATTGGATGGTAAGATAATTACGCTAAATCACTCCATACTGGA
TTTGAAGTCACATATTCTGATCAAGCGTTCTCAATGTCCCACCTGTGGTGACCC
AAAAATCTTACAGCACCGTGGTTTCGAACCTTTAAAACTTGAGTCAAGGCCTAA
ACAGTTCACCTCAGACGGCGGACATCGTGGTACTACCCCTGAACAAACTGTCCA
GAAATATCAACATTTAATCTCGCCTGTTACCGGTGTAGTTACTGAATTGGTCAG
GATAACTGATCCGGCCAATCCACTAGTTCACACATATAGAGCTGGTCATAGCTT
CGGGAGCGCTACATCGCTGAGAGGGCTGCGTAATACCTTAAAGCATAAGAGTTC
AGGTAAGGGTAAGACTGATTCTCAAAGTAAAGCCTCGGGCCTGTGTGAGGCGGT
AGAACGTTACTCAGGAATCTTTCAAGGTGACGAACCGAGAAAACGCGCCACATT
GGCTGAATTGGGAGATTTGGCAATTCACCCTGAGCAATGCTTGTGTTTTTCCGA
CGGTCAGTACGCTAATAGAGAAACTTTAAACGAACAGGCAACGGTGGCACATGA
TTGGATACCTCAACGTTTTGATGCATCACAAGCTATTGAATGGACTCCAGTCTG
GTCCCTAACTGAACAGACCCATAAATATTTGCCCACCGCATTGTGTTACTACCA
TTATCCTCTACCCCCAGAACACAGATTCGCACGTGGAGATTCGAATGGTAATGC
TGCCGGAAATACGTTGGAAGAGGCTATACTCCAAGGCTTCATGGAATTAGTCGA
GAGAGATGGTGTGGCTTTATGGTGGTATAACAGGCTACGCAGACCCGCTGTAGA
CTTAGGCTCATTTAACGAGCCATACTTCGTTCAGTTGCAACAATTCTACAGAGA
AAACGATAGAGATTTGTGGGTTTTGGACTTGACAGCTGATTTAGGTATCCCGGC
TTTCGCGGGCGTTTCTAATAGAAAAACTGGTAGTTCGGAGAGGTTGATATTAGG
ATTCGGTGCACACCTCGATCCTACTATTGCAATTCTGAGAGCAGTTACAGAAGT
TAACCAGATTGGCCTTGAATTAGATAAAGTTCCAGACGAGAACCTTAAGAGCGA
CGCAACAGATTGGCTAATTACTGAAAAATTAGCTGACCACCCTTATTTGTTACC
AGATACAACTCAACCTCTAAAAACTGCTCAAGATTATCCTAAAAGGTGGTCTGA
CGATATATACACGGACGTAATGACTTGCGTTAATATTGCTCAACAAGCAGGACT
TGAAACTCTAGTTATTGATCAAACACGTCCGGACATTGGTTTGAATGTTGTTAA
GGTGACAGTCCCGGGGATGAGGCACTTTTGGTCAAGATTTGGAGAGGGGAGGCT
TTATGACGTGCCCGTCAAATTAGGTTGGCTTGACGAACCTTTGACCGAAGCGCA
AATGAACCCCACGCCGATGCCTTTTTAATAA
ACCAAATACATCGCGTTTGGTCTGCGCATTGCCAGCGAACTCAACTTACCGGAA
CTGATATTGGCGGCTCCCGAAGCCGTTGAGGATGTTGTCATACGCCAGGCAGAT
CTCACGGCCTGGTCTGGCCAACTTGAACAGGCAAATTTTGTCATGTTGGACGAA
CGTTTCATGTTTCAGATCCCGGGGACCGCCATTTATGCGGTACGCGAAGGCAAA
GAGATTGAAGTGAGCATCTTCTCTGGGGCCGACCCGGACACCGTGCGCCTTTTC
GTGCTGGGGACGTGCATGGGCGTGCTCTTGATGCAGCGCCGCATTCTGCCTATC
CACGGCTCCGCCGTCGTTATCGGTGGCCGCGCGTATGCCTTTGTTGGTGAATCA
GGCACAGGTAAATCGACCTTAGCTGCAGCATTTCGGCAGGCCGGTTACCAAATG
GTTAGCGATGATGTCATTGCCGTCAAAGCGACCGCATCTAGCGCTATTGTTTAC
CCTGCGTATCCACAGCAAAAACTGGGTTTAGATTCGCTGTTGCAGCTTGAAGCG
CTCCGTGAGAATAAGCACGCCCGCAAGCGTAACAACATCCGTTCTCTGACGGAT
GGCAATAGTGTGATGCCGCAGTACAGCGATCTGCGCATGCTGGCGGGGGAACTG
AATAAATATGCAGTTCCAGCCGTCGATGAATTCTTTAATGACCCGCTGCCGTTG
GGCGGTGTTTTCGAACTGGTAGCAGACAGTCCGATTCGAGCATTAATGCGCGAA
GGCGAACTCGTCGCTGTGACCGAGCAACCGCTGAACGTTCTGGAATGTTTACAT
ACTCTTCTGCAACACACGTACCGTCGGGTAATCATCCCTCGAATGGGACTGAGC
GAGTGGAGCTTCGATACTGCGGCCCGAATGGCACGCAAGGTCGAGGGCTGGCGA
CTCCTCCGTGATAGCTCCGTGTTCACGGCTAGTGAAGTCGTCCAGCGCGTCCTC
GACATCATCCGTAAGGAGGAAAAGAGCTACGGATCACACTAATAA
GCTTCGATCAACGTCATCAATATCAACCATTATATTGTGGAGCTGAAACAGCAC
TTCGATGAGGTGAATATCCTGTTTTCACCTTCCTCGAAGAACTTTATCAACACC
GATGTCCTGAAGCTGTTTTGCGATAATCTGTATGACGAGATCAAAGATCCGCTG
CTGAACCACATCAACATAGTGGAGAACCACGAGTATATCTTGGTGCTGCCTGCC
AGTGCCAATACGATCAACAAAATCGCGAACGGTATATGCGATAACCTCTTGACG
ACCGTATGCTTAACCGGGTACCAGAAACTGTTTATCTTTCCGAATATGAACATC
CGCATGTGGGGAAATCCGTTCTTACAGAAAAATATTGACCTGCTTAAAAGCAAC
GACGTGAAGGTGTATTCCCCCGACATGAACAAATCTTTTGAGATAAGCTCAGGC
CGCTACAAAAATAACATCACGATGCCGAATATCGAAAACGTGCTGAATTTTGTC
CTGAACAATGAGAAACGCCCGCTGGATTAATAA
AAGTGCATAGTCGTATACACAAACTGCAAAATAATATCGCAATAGGTAGCATGC
CGCCTCACGCGCTGATCATCGAGGATGCCCCCGAATATTTGTCAAACGTTCTGC
GCTTCTTTAGTAGCAAAAAGACTATAAAAGAAGCTGAAGTGTACCTGTCGGATA
ATACGAATCTGAGCTCCAATGAGATCAACCTGTTGTTAGGTGATCTGATTGAGA
ACGAGATTATCGTAAAGCAAAACTACGACTCGAATAATCGGTACAGTCGACACA
GTCTGTATTACGAGATGATTGATGCCAACGCTGAAAACGCGCAGAAAATTCTGG
CAGAGAAAACAGTGGGCCTCGTTGGGATGGGCGGGATTGGTTCCAATGTAGCCA
TGAATCTCGCAGCCGCCGGTGTTGGCAAACTGATCTTTAGTGATGGCGATACCA
TAGAACTGTCTAATTTAACGCGACAGTATCTTTACAAAGAGGATCAGGTGGGCT
TGAGCAAAGTAGAGAGCGCCAAAGAACAACTGCAATTACTGAATAGCGAAGTCG
AGCTTATCCCGGTTTGCGAAAGTATCTCTGGTGAGGAACTGTTCGACAACCATT
TCTCCGAATGCGATTTCGTCGTACTGTCCGCCGACTCTCCGTTCTTTGTTCACG
AATGGATTAACAATGCCGCGTTGAAATATGGCTTCTCCTACTCTAACGCAGGAT
ATATCGAAACCTATGGCGCGATCGGTCCACTGGTGATACCTGGGGAAACTGCCT
GCTACGAATGCTATAAAGACAAGGGCGATCTTTACTTGTACTCCGACAACAAGG
AAGAATTTTCTGTGAACCTGAATGAATCATTCCAAGCACCGAGCTATGGACCGC
TTAATGCGATGGTTAGTTCCATTCAGGCGAATGAAGTGATACGCCACCTCCTCG
GACTTAAAACCAAAACGTCCGGCAAACGGCTGCTGATCAACAGTGAAATCTACA
AAATCCACGAAGAGAACTTCGAGAAGAAGAACAACTGCCTGTGCTCGGATATTA
AGGGCGAGAAGCTGTCGAAGAACACCCTTAACTCCGATAAAGAGCTGCACGAAG
TGTATATCGAAGAACGCGAATCGGATTCTTTCAACTCCATTCTCTTGGATAAAA
CCATGAGCAAGCTGGTAAAAATTAACAAAGAGGAGACAAAAATCCTCGACATTG
GTTGCGCTACCGGCGAACAGGCTCTGTATTTCGCGAATAAAGGTGCTAAGGTGA
CCGCTGTCGACATTTCAGACGATATGTTGAAGGTGCTGGACAAGAAAGCAAGCA
ACATTAACGCGGGGAGTATCAAAACCATGCGTGGTAATATCGAATCCATCGAGG
TGAATGACACTTTTAATTACATCGTCTGTAACAACATCCTTGATTACCTGCCGG
AGATCGACCGCACGCTGAGAAAACTTAACATGTTTTTGAAAAATGACGGGACGC
TGATTGTGACGATTCCCCACCCCGTGAAGGATGGTGGAGGGTGGCGGAAAGATT
ATTATAACGGCAAATGGAACTACGAAGAGTTTATCCTGAAGGATTACTTCAACG
AGGGTCTGATCGAAAAGAGCCGCGAGGACAAAAATGGGGAAACGGTGATCAAAA
GCATTAAAACGTACCACAGAACCACCGAAACCTATTTCAATAGCTTTACTGACG
CTGGCTTCAAGGTAGTATCTCTGCTGGAACCGCAACCGCTTTCAACTGTTTCAG
AGACTCATCCAATTCTGTTCGAAAAGTGTTCGCGCATTCCGTACTTTCAAGTTT
TTGTGCTCAAGAAAGAGGATCGCCACGCCATTTAATAA
aIn each backbone sequence (labeled “bEG_SX”, where X is a number), the relevant part (encoding a peptide or RBS + enzyme) has GFP as a placeholder (RBS + GFP for enzyme plasmid) and is double underlined. This region can be replaced with an insert DNA (such as a peptide or RBS + enzyme, including those listed below each plasmid backbone sequence) to get a plasmid sequence. The full plasmid sequences used herein (labeled “pEG####”) for peptides and enzymes can be identified by replacing the double underlined portion of the backbone sequence found above a given peptide/RBS + enzyme sequence with the respective peptide/RBS + enzyme sequence (for example, the full pEG3045 plasmid sequence is provided by replacing the double underlined portion of the bEG_S2 backbone sequence with the HIS6-MdnA provided next to the pEG3045 label).
bText is formatted according to sequence components: promoters (lowercase), ribozyme
(UNDERLINED), and plasmid backbone and spacers (REGULAR ALL CAPS).
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 63/038,394, filed Jun. 12, 2020, which is incorporated by reference herein in its entirety.
This invention was made with Government support under Grant No. HR0011-15-C-0084 awarded by the Defense Advanced Research Projects Agency (DARPA). The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/037120 | 6/11/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63038394 | Jun 2020 | US |
