EXPRESSION VECTORS FOR EUKARYOTIC EXPRESSION SYSTEMS

FIELD OF THE INVENTION

The present invention relates to expression vectors for expressing recombinant proteins (e.g., biologics or vaccines) in eukaryotic cells.

BACKGROUND OF THE INVENTION

Most biopharmaceuticals are produced in mammalian cells transfected with an expression vector that drives constitutive and high-level expression of the recombinant protein (See, e.g., Wurm, F. M., Nature Biotech. 22:1393-1398 (2004)). Chinese hamster ovary (CHO) cell line is one of the most commonly used cell lines in the commercial production of recombinant protein therapeutics, including monoclonal antibodies. Increased demand for protein therapeutics has bolstered efforts to augment cell line productivity through improvements in expression technology and optimization of process conditions. (See, e.g., Wurm, supra; Birch, J. R. & Racher, A. J., Adv. Drug Delivery Rev. 58:671-685 (2006)).

A well-designed expression vector is the first step toward achieving high production of recombinant proteins. (See, e.g., Ludwig, D. L., BioProcess International 4:S14-S23 (2006)).

Expression vectors generally include a number of components, for example, one or more polypeptide expression cassettes, one or more selectable markers, and elements that allow replication of the vector in prokaryotic cells. In general, selection of the different components to be included in an expression vector will impact target polypeptide expression in mammalian host cells, and it is typically unpredictable if any new combination of components will support high levels of polypeptide expression.

SUMMARY OF THE INVENTION

The present disclosure provides expression vectors for eukaryotic expression systems that can 1) efficiently integrate into eukaryotic transcriptionally active hot spots; 2) block epigenetic gene silencing to ensure long term stable expression; 3) link the gene of interest (GOI) and the eukaryotic selectable marker to ensure consistent expression of GOI in the surviving eukaryotic cells; 4) remain stable to support various processes, including continuous perfusion; and 5) support faster timelines from construction to final clone selection.

In one aspect, provided herein is an expression vector comprising:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a promoter operably linked to an insertion site for a GOI, an internal ribosome entry site (IRES), a polynucleotide encoding a eukaryotic selectable marker, and a polyadenylation (polyA) signal;

(b) a second expression cassette comprising a polynucleotide encoding a bacterial selectable marker; and

In certain embodiments of the various expression vectors provided herein, the first expression cassette further comprises one or more regulatory elements. In some embodiments, the regulatory element is an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), or a ubiquitous chromatin opening element (UCOE). In one embodiment, the regulatory element is an enhancer. In another embodiment, the regulatory element is an insulator. In yet another embodiment, the regulatory element is a LCR. In still another embodiment, the regulatory element is a MAR. In one embodiment, the regulatory element is a SAR. In another embodiment, the regulatory element is an EASE. In yet another embodiment, the regulatory element is a TPL. In still another embodiment, the regulatory element is a UCOE. In some embodiments, the first expression cassette further comprises one regulatory element. In other embodiments, the first expression cassette further comprises two regulatory elements. In yet other embodiments, the first expression cassette further comprises three regulatory elements. In still other embodiments, the first expression cassette further comprises four regulatory elements. In some embodiments, the first expression cassette further comprises five regulatory elements. In other embodiments, the first expression cassette further comprises six regulatory elements. In yet other embodiments, the first expression cassette further comprises seven regulatory elements. In still other embodiments, the first expression cassette further comprises eight or more regulatory elements.

In some embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette. In other embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises one or more regulatory elements. In yet other embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises one or more regulatory elements selected from the group consisting of an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), and a ubiquitous chromatin opening element (UCOE). In certain embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises one regulatory element selected from the group consisting of an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), and a ubiquitous chromatin opening element (UCOE). In some embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises two regulatory elements selected from the group consisting of an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), and a ubiquitous chromatin opening element (UCOE). In other embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises three regulatory elements selected from the group consisting of an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), and a ubiquitous chromatin opening element (UCOE). In yet other embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises four regulatory elements selected from the group consisting of an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), and a ubiquitous chromatin opening element (UCOE). In still other embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises five regulatory elements selected from the group consisting of an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), and a ubiquitous chromatin opening element (UCOE). In some embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises six regulatory elements selected from the group consisting of an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), and a ubiquitous chromatin opening element (UCOE). In other embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises seven regulatory elements selected from the group consisting of an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), and a ubiquitous chromatin opening element (UCOE). In yet other embodiments, the expression vector further comprises two inverted terminal repeat (ITR) sequences flanking the first expression cassette, and the first expression cassette further comprises eight regulatory elements selected from the group consisting of an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), and a ubiquitous chromatin opening element (UCOE).

In some embodiments of the various expression vectors provided herein, the eukaryotic selectable marker is a neomycin phosphotransferase, a histidinol dehydrogenase, a hygromycin B phosphotransferase, a xanthine-guanine phosphoribosyltransferase, a dihydrofolate reductase, a tryptophan synthetase, a puromycin N-acetyl-transferase, a thymidine kinase, an adenine phosphoribosyl transferase, a glutamine synthetase, an adenosine deaminase, or metallothionein-1. In one embodiment, the eukaryotic selectable marker is a neomycin phosphotransferase. In another embodiment, the eukaryotic selectable marker is a histidinol dehydrogenase. In yet another embodiment, the eukaryotic selectable marker is a hygromycin B phosphotransferase. In still another embodiment, the eukaryotic selectable marker is a xanthine-guanine phosphoribosyltransferase. In one embodiment, the eukaryotic selectable marker is a dihydrofolate reductase. In another embodiment, the eukaryotic selectable marker is a tryptophan synthetase. In yet another embodiment, the eukaryotic selectable marker is a puromycin N-acetyl-transferase. In still another embodiment, the eukaryotic selectable marker is a thymidine kinase. In one embodiment, the eukaryotic selectable marker is an adenine phosphoribosyl transferase. In another embodiment, the eukaryotic selectable marker is a glutamine synthetase. In yet another embodiment, the eukaryotic selectable marker is an adenosine deaminase. In still another embodiment, the eukaryotic selectable marker is metallothionein-1.

In certain embodiments of the various expression vectors provided herein, the promoter is a human cytomegalovirus (CMV) immediate-early promoter, a human elongation factor 1 alpha (EF1a) promoter, a SV40 promoter, a phosphoglycerate kinase 1 (PGK1) promoter, a human ubiquitin C (Ubc) promoter, a human β-actin promoter, a CAG promoter, a yeast transcription elongation factor 1 (TEF1) promoter, a yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or a yeast alcohol dehydrogenase 1 (ADH1) promoter. In one embodiment, the promoter is a human CMV immediate-early promoter. In another embodiment, the promoter is a human EFla promoter. In yet another embodiment, the promoter is a SV40 promoter. In still another embodiment, the promoter is a PGK1 promoter. In one embodiment, the promoter is a human Ubc promoter. In another embodiment, the promoter is a human β-actin promoter. In yet another embodiment, the promoter is a CAG promoter. In still another embodiment, the promoter is a yeast TEF1 promoter. In one embodiment, the promoter is a yeast GAPDH promoter. In another embodiment, the promoter is a yeast ADH1 promoter.

In some embodiments of the various expression vectors provided herein, the enhancer is a human CMV immediate-early enhancer, a SV40 enhancer, a BK polyomarvirus (BKPyV) enhancer, an Epstein-Bar virus (EBV) enhancer, a c-Myc enhancer, an immunoglobulin heavy chain (IgH) enhancer, a Sp1-binding enhancer, an AP1-binding enhancer, or a CREB-binding enhancer. In one embodiment, the enhancer is a human CMV immediate-early enhancer. In another embodiment, the enhancer is a SV40 enhancer. In yet another embodiment, the enhancer is a BKPyV enhancer. In still another embodiment, the enhancer is an EBV enhancer. In one embodiment, the enhancer is a c-Myc enhancer. In another embodiment, the enhancer is an IgH enhancer. In yet another embodiment, the enhancer is a Sp1-binding enhancer. In still another embodiment, the enhancer is an AP1-binding enhancer. In one embodiment, the enhancer is a CREB-binding enhancer.

In certain embodiments, the enhancer is immediately adjacent to the promoter. In some embodiments, the enhancer is distant from the promoter with other DNA fragments between the enhancer and the promoter. In other embodiments, the enhancer is upstream of the promoter. In yet other embodiments, the enhancer is downstream of the promoter. In still other embodiments, the enhancer and the promoter are combined together as a combo enhancer/promoter. In one specific embodiment, the combo enhancer/promoter is a human CMV immediate-early enhancer/promoter. In another specific embodiment, the combo enhancer/promoter is a synthetic CAG promoter that comprises a CMV immediate-early enhancer and a chicken β-actin promoter.

In other embodiments of the various expression vectors provided herein, the insulator is HMR tRNA^Thr, Chal UAS, UAS_rpg, STAR, scs, scs′, gypsy, Fab-7, Fab-8, fas^wb, sns, UR1, RO, Lys 5′ A, HS4, 3′HS, BEAD-1, HS2-6, DMD/ICR, 5′HS5, apoB (−57 kb), apoB (+43 kb), or DM1. In one embodiment, the insulator is HMR tRNA^Thr. In another embodiment, the insulator is Chal UAS. In yet another embodiment, the insulator is UAS_rpg. In still another embodiment, the insulator is STAR. In one embodiment, the insulator is scs. In another embodiment, the insulator is scs′. In yet another embodiment, the insulator is gypsy. In still another embodiment, the insulator is Fab-7. In one embodiment, the insulator is Fab-8. In another embodiment, the insulator is fas^wb. In yet another embodiment, the insulator is sns. In still another embodiment, the insulator is UR1. In one embodiment, the insulator is RO. In another embodiment, the insulator is Lys 5′ A. In yet another embodiment, the insulator is HS4. In still another embodiment, the insulator is 3′HS. In one embodiment, the insulator is BEAD-1. In another embodiment, the insulator is HS2-6. In yet another embodiment, the insulator is DMD/ICR. In still another embodiment, the insulator is 5′HS5. In one embodiment, the insulator is apoB (−57 kb). In another embodiment, the insulator is apoB (+43 kb). In yet another embodiment, the insulator is DM1.

In certain embodiments of various expression vectors provided herein, the first expression cassette further comprises a first insulator and a second insulator. In some embodiments, the first insulator and the second insulators are the same insulator. In some embodiments, the first insulator and the second insulator are different insulators. In other embodiments, the first insulator and the second insulator are in the same direction. In yet other embodiments, the first insulator and the second insulator are in the opposite directions. In still other embodiments, the first insulator and the second insulator are HS4. In yet still other embodiments, the first insulator and the second insulator are HS4 in the opposite directions.

In yet other embodiments of the various expression vectors provided herein, the ITR is Tc1 ITR, Tc3 ITR, Minos ITR, Mos1 ITR, Famar1 ITR, Osmar5 ITR, Fot1 ITR, Impala ITR, ISY100 ITR, Mboumar-9 ITR, Sleeping Beauty ITR, Himar1 ITR, Frog Prince ITR, Hsmar1 ITR, SB100X ITR, piggyBac ITR, or Tol2 ITR. In one embodiment, the ITR is Tc1 ITR. In another embodiment, the ITR is Tc3 ITR. In yet another embodiment, the ITR is Minos ITR. In still another embodiment, the ITR is Mos1 ITR. In one embodiment, the ITR is Famar1 ITR. In another embodiment, the ITR is Osmar5 ITR. In yet another embodiment, the ITR is Fot1 ITR. In still another embodiment, the ITR is Impala ITR. In one embodiment, the ITR is ISY100 ITR. In another embodiment, the ITR is Mboumar-9 ITR. In yet another embodiment, the ITR is Sleeping Beauty ITR. In still another embodiment, the ITR is Himar1 ITR. In one embodiment, the ITR is Frog Prince ITR. In another embodiment, the ITR is Hsmar1 ITR. In yet another embodiment, the ITR is SB100XITR. In still another embodiment, the ITR is piggyBac ITR. In one embodiment, the ITR is Tol2 ITR.

In certain embodiments of the various expression vectors provided herein, the bacterial selectable marker is an ampicillin resistance gene, a tetracycline resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a blasticidin resistance gene, or the like. In one embodiment, the bacterial selectable marker is an ampicillin resistance gene. In another embodiment, the bacterial selectable marker is a tetracycline resistance gene. In yet another embodiment, the bacterial selectable marker is a hygromycin resistance gene. In still another embodiment, the bacterial selectable marker is a kanamycin resistance gene. In yet still another embodiment, the bacterial selectable marker is a blasticidin resistance gene.

In another aspect, provided herein is an expression vector comprising:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first insulator, an EASE, a promoter, a TPL, an insertion site for a GOI, an IRES, a polynucleotide encoding a eukaryotic selectable marker, a polyA signal, and a second insulator;

(b) two ITR sequences flanking the first expression cassette;

(d) a bacterial plasmid origin of replication.

In some embodiments of various expression vectors provided herein, the first expression cassette further comprises an enhancer. In certain embodiments, the enhancer is located between the EASE and the promoter.

In other embodiments of the various expression vectors provided herein, the insulator is HMR tRNA^Thr, Chal UAS, UAS_ipg, STAR, scs, scs′, gypsy, Fab-7, Fab-8, fas^wb, sns, UR1, RO, Lys 5′ A, HS4, 3′HS, BEAD-1, HS2-6, DMD/ICR, 5′HS5, apoB (−57 kb), apoB (+43 kb), or DM1. In one embodiment, the insulator is HMR tRNA^Thr. In another embodiment, the insulator is Chal UAS. In yet another embodiment, the insulator is UAS_rpg. In still another embodiment, the insulator is STAR. In one embodiment, the insulator is scs. In another embodiment, the insulator is scs′. In yet another embodiment, the insulator is gypsy. In still another embodiment, the insulator is Fab-7. In one embodiment, the insulator is Fab-8. In another embodiment, the insulator is fas^wb. In yet another embodiment, the insulator is sns. In still another embodiment, the insulator is UR1. In one embodiment, the insulator is RO. In another embodiment, the insulator is Lys 5′ A. In yet another embodiment, the insulator is HS4. In still another embodiment, the insulator is 3′HS. In one embodiment, the insulator is BEAD-1. In another embodiment, the insulator is HS2-6. In yet another embodiment, the insulator is DMD/ICR. In still another embodiment, the insulator is 5′HS5. In one embodiment, the insulator is apoB (−57 kb). In another embodiment, the insulator is apoB (+43 kb). In yet another embodiment, the insulator is DM1.

In some embodiments, the first insulator and the second insulators are the same insulator. In some embodiments, the first insulator and the second insulator are different insulators. In other embodiments, the first insulator and the second insulator are in the same direction. In yet other embodiments, the first insulator and the second insulator are in the opposite directions. In still other embodiments, the first insulator and the second insulator are HS4. In yet still other embodiments, the first insulator and the second insulator are HS4 in the opposite directions.

In one embodiment of various expression vectors provided herein, the first insulator and the second insulator are HS4, the enhancer is a human CMV immediate-early enhancer, the promoter is a human CMV immediate-early promoter, the ITR is piggyBac ITR, the eukaryotic selectable marker is a glutamine synthetase, and the IRES comprises a polynucleotide sequence of SEQ ID NO:3.

In another embodiment of various expression vectors provided herein, the first insulator and the second insulator are HS4, the enhancer and the promoter are a combo enhancer/promoter, wherein the combo enhancer/promoter is a human CMV immediate-early enhancer/promoter, the ITR is piggyBac ITR, the eukaryotic selectable marker is a glutamine synthetase, and the IRES comprises a polynucleotide sequence of SEQ ID NO:3.

Thus, in one particular embodiment, the expression vector comprises:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first HS4 insulator, an EASE, a human CMV immediate-early enhancer/promoter, a TPL, an insertion site for a GOI, an IRES comprising a polynucleotide sequence of SEQ ID NO:3, a polynucleotide encoding a glutamine synthetase, a polyA signal, and a second HS4 insulator;

(b) two piggyBac ITR sequences flanking the first expression cassette;

(d) a bacterial plasmid origin of replication.

In another specific embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 26, 27, 28, or 29. In one embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:4. In another embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:5. In yet another embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:6. In still another embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:7. In yet still another embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:8. In one embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:26. In another embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:27. In yet another embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:28. In still another embodiment, the expression vector comprises a polynucleotide sequence of SEQ ID NO:29.

In yet another specific embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 26, 27, 28, or 29. In one embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO:4. In another embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO:5. In yet another embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO:6. In still another embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO:7. In yet still another embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO:8. In one embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO:26. In another embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO:27. In yet another embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO:28. In still another embodiment, the expression vector consists of a polynucleotide sequence of SEQ ID NO:29.

In yet still another specific embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 26, 27, 28, or 29. In one embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO:4. In another embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO:5. In yet another embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO:6. In still another embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO:7. In yet still another embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO:8. In one embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO:26. In another embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO:27. In yet another embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO:28. In still another embodiment, the expression vector consists essentially of a polynucleotide sequence of SEQ ID NO:29.

In another specific embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 26, 27, 28, or 29. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:4. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:5. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:6. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:7. In yet still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:8. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:26. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:27. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:28. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 60%, 70%, 80%, 90%, or 95% identical to the polynucleotide sequence of SEQ ID NO:29.

In certain embodiments of the various expression vectors provided herein, the expression vector further comprises the GOI. In some embodiments, the expression vector further comprises the GOI, and the GOI encodes a therapeutic or prophylactic protein. In other embodiments, the expression vector further comprises the GOI, and the GOI encodes a heavy chain or a fragment thereof of a monoclonal antibody. In yet other embodiments, the expression vector further comprises the GOI, and the GOI encodes a light chain or a fragment thereof of a monoclonal antibody.

In yet another aspect, provided herein is a mammalian recombinant host cell comprising a mammalian host cell transfected with the expression vector described herein.

In still another aspect, provided herein is a method of producing a polypeptide, comprising culturing the mammalian recombinant host cell described herein, under conditions in which the polypeptide is expressed.

In some embodiments, provided herein is a method of producing a polypeptide, comprising culturing the mammalian recombinant host cell described herein, under conditions in which the polypeptide is expressed, and recovering the polypeptide from the culture.

In another aspect, provided herein is a bacterial recombinant host cell comprising a bacterial host cell transformed with the expression vector described herein.

In yet another aspect, provided herein is a method of propagating an expression vector, comprising culturing the bacterial recombinant host cell described herein, under conditions in which the expression vector is replicated.

In some embodiments, provided herein is a method of propagating an expression vector, comprising culturing the bacterial recombinant host cell described herein, under conditions in which the expression vector is replicated, and recovering the expression vector from the culture.

In still another aspect, provided herein is a mammalian recombinant host cell comprising a mammalian host cell co-transfected with a first expression vector and a second expression vector, wherein the first expression vector and the second expression vector are each expression vectors described herein, wherein the GOI of the first expression vector encodes a light chain of a monoclonal antibody and the GOI of the second expression vector encodes a heavy chain of the monoclonal antibody, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In yet still another aspect, provided herein is a method of producing a monoclonal antibody, comprising culturing the mammalian recombinant host cell co-transfected with a first expression vector described herein comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector described herein comprising a second GOI encoding the heavy chain of the monoclonal antibody, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed.

In some embodiments, provided herein is a method of producing a monoclonal antibody, comprising culturing the mammalian recombinant host cell co-transfected with a first expression vector described herein comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector described herein comprising a second GOI encoding the heavy chain of the monoclonal antibody, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and recovering the monoclonal antibody from the culture.

In certain embodiments, the method of producing a monoclonal antibody comprises culturing the mammalian recombinant host cell co-transfected with a first expression vector described herein comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector described herein comprising a second GOI encoding the heavy chain of the monoclonal antibody, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In other embodiments, the method of producing a monoclonal antibody comprises culturing the mammalian recombinant host cell co-transfected with a first expression vector described herein comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector described herein comprising a second GOI encoding the heavy chain of the monoclonal antibody, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and recovering the monoclonal antibody from the culture, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary expression vector with engineered elements. E represents an enhancer; P represents a promoter; ORF represents an open reading frame; UTR represents an untranslated region; IRES represents an internal ribosome entry site; PolyA represents a polyadenylation signal; ITR represents an inverted repeat region, Bac-Ori represents a bacterial origin of replication; Amp represents an ampicillin selectable marker.

FIGS. 2A-2E illustrate the structure of exemplary expression vectors with GOI: pCLD116-GOI (FIG. 2A), pCLD21-GOI (FIG. 2B), pCLD22-GOI (FIG. 2C), pCLD12-HOI (FIG. 2D), and pCLD14-GOI (FIG. 2E).

FIG. 3 shows that the expression vectors using transposon technology significantly reduced selection time for stable expressing cells.

FIG. 4 shows that the shortened selection time for stable expressing cells is independent of host cell lines.

FIG. 5 demonstrates that high expression of a monoclonal antibody was achieved by engineering expression vectors with appropriate configurations.

FIGS. 6A-6D demonstrate the effect of the nucleotide length of IRES on titer (FIG. 6A), specific productivity (FIG. 6B), aggregate level (FIG. 6C), and mannose level (FIG. 6D) of a monoclonal antibody production.

FIGS. 7A and 7B demonstrate the effect of the nucleotide length of IRES on DNA copy number (FIG. 7A) and mRNA level (FIG. 7B) of GOI encoding a monoclonal antibody heavy chain or light chain.

DETAILED DESCRIPTION OF THE INVENTION
I. General

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosure of such documents are incorporated herein by reference in their entirety for all purposes, and to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

II. Molecular Biology and Definitions

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook, et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this specification, all other technical and scientific terms use herein have the meaning that would be commonly understood by one of ordinary skill in the art to which this invention belongs when used in similar contexts as used herein.

As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.

“About” when used to modify a numerically defined parameter, e.g., the length of a polynucleotide discussed herein, means that the parameter may vary by as much as 10% below or above the stated numerical value for that parameter. For example, a polynucleotide of about 100 bases may vary between 90 and 110 bases.

A “coding sequence” is a nucleotide sequence that encodes a biological product of interest (e.g., an RNA, polypeptide, protein, or enzyme) and when expressed, results in production of the product. A coding sequence is “under the control of,” “functionally associated with,” “operably linked to,” or “operably associated with” transcriptional or translational regulatory sequences in a cell when the regulatory sequences direct RNA polymerase-mediated transcription of the coding sequence into RNA, e.g., mRNA, which then may be trans-RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.

“Consists essentially of” and variations such as “consist essentially of” or “consisting essentially of” as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, which do not materially change the basic or novel properties of the specified composition.

“Express” and “expression” mean allowing or causing the information in a gene or coding sequence, e.g., an RNA or DNA, to become manifest; for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene. A DNA sequence can be expressed in or by a cell to form an “expression product” such as an RNA (e.g., mRNA) or a protein. The expression product itself may also be said to be “expressed” by the cell.

“Expression vector” or “expression construct” means a vehicle (e.g., a plasmid) by which a polynucleotide comprising regulatory sequences operably linked to a coding sequence can be introduced into a host cell where the coding sequence is expressed using the transcription and translation machinery of the host cell.

“Expression cassette” means a polynucleotide that comprises elements sufficient to control expression of a gene, including but not limited to, a promoter operably linked to the gene sequence or operably linked to a multiple cloning site for inserting the gene sequence, and a polyA signal. In some embodiments, the expression cassette further comprises one or more regulatory elements that can regulate the expression of the gene at transcriptional, translational, and/or chromatin levels.

“Promoter” or “promoter sequence” is a segment of DNA that contains a regulatory region capable of recruiting an RNA polymerase (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the recruiting of RNA polymerase.

“Enhancer” or “enhancer sequence” is a DNA regulatory region that enhances transcription of a promoter independently of its distance, location, or orientation to the promoter. In certain embodiments, the enhancer is immediately adjacent to the promoter. In some embodiments, the enhancer is distant from the promoter. In other embodiments, the promoter and the enhancer are one combined sequence, referred as a “combo enhancer/promoter” herein.

“Internal ribosome entry site” or “IRES” is an RNA element or sequence that allows for translation initiation in a cap-independent manner by recruiting ribosomes directly. As used herein, the term “internal ribosome entry site” or “IRES” also encompasses the DNA sequence that can be transcribed into the RNA sequence that allows for translation initiation in a cap-independent manner by recruiting ribosomes directly. IRES can be a wild type IRES from any species or a variant or mutant thereof, whether naturally occurred or man-made. Examples of IRES that can be used include, but are not limited to, the nucleotide sequence of the 5′ nontranslated region of encephalomyocarditis virus (EMCV) (GenBank: M81861.1; Duke et al., Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. J Virol. 1992 March; 66(3):1602-9.), IRES element described by Bochkov & Palmenberg (Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques. 2006 September; 41(3):283-4), IRES element from expression vector pInSRT-GFP (GenBank LC417349.1), IRES element from expression vector pCeMM-CTAP(SG) (GenBank EF467048.1), IRES element described by Jang & Wimmer (Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 September; 4(9):1560-72), IRES element from expression vector pIRESneo3 (Clontech/Takara Bio), IRES elements described in WO 2015/016786, WO 2015/021077, WO 2016/003368, WO 2016/074016, or WO 2013/092743, or variants thereof.

“Regulatory element,” “regulatory region,” or “regulatory sequence,” as used herein, refers to a polynucleotide sequence that has the ability to regulate (such as, initiate, activate, enhance, increase, decrease, inhibit, suppress, or silence) expression of a gene. In some embodiments, the regulation is achieved by binding of cellular factors to the polynucleotide sequence. In other embodiments, the regulation is achieved by interaction between cellular factors. The regulation can occur at one or more different levels in the expression process from DNA to protein, including but not limited to transcriptional, translational, or chromatin levels.

“Insulator,” as used herein, refers to a class of DNA elements or sequences that possess an ability to isolate the proximal DNA region by preventing the positional effect from the surrounding chromosome area. In certain embodiments, the insulator can block enhancer when the insulator is situated between the enhancer and the promoter. In some embodiments, the insulator can act as barriers that prevent the advance of nearby condensed chromatin that might otherwise silence expression. In other embodiments, the insulator can block enhancer and act as barriers.

“Expression augmenting sequence element” or “EASE” is a DNA element or sequence that can increase expression of a protein when the DNA element or sequence is placed upstream of the promoter that controls the expression of the protein.

“Tripartite leader” or “TPL” is an RNA element or sequence in the 5′-untranslated region of adenovirus late-expressed mRNA that has an ability to initiate translation of the late-expressed mRNA in a cap-independent manner. As used herein, the term “tripartite leader” or “TPL” also encompasses the DNA sequence that can be transcribed into the RNA sequence in the 5′-untranslated region of adenovirus late-expressed mRNA that has an ability to initiate translation of the late-expressed mRNA in a cap-independent manner.

“Inverted terminal repeat” or “ITR,” in the context of transposon technology, refers to a DNA element or sequence and its inverted version at either end of a transposon that signals where the breakage and joining should occur.

“Selectable marker” or “selection marker” is a protein which allows the specific selection of cells that express this protein by the addition of a corresponding selecting agent to the culture medium. In certain embodiments, the selectable marker is a eukaryotic selectable marker, which allows selection of eukaryotic cells that express the marker protein. In some embodiments, the selectable marker is a bacterial selectable marker, which allows selection of bacterial cells that express the marker protein.

“Nucleic acid” or “polynucleotide” refers to a single- or double-stranded polymer of bases attached to a sugar phosphate backbone, and includes DNA and RNA molecules.

Each strand of DNA or RNA has a 5′ end and a 3′ end. “Direction,” as used herein, when referring to a DNA, means the 5′ to 3′ direction of the coding strand for a gene, and, when referring to an RNA, means the 5′ to 3′ direction of the RNA molecule. When two DNA or RNA fragments are in the “same direction,” their 5′ to 3′ directions align and are in the same direction. When two DNA or RNA fragments are in the “opposite direction,” their 5′ to 3′ directions are opposite.

“Upstream” or “downstream,” as used herein, means relative positions of nucleic acid in DNA when referring to a gene or in RNA when referring to a gene transcript. When referring to the 5′ to 3′ direction in which RNA transcription takes place, upstream is toward the 5′ end of the RNA molecule and downstream is toward the 3′ end of the RNA. When referring to a double-stranded DNA, upstream is toward the 5′ end of the coding strand for the gene and downstream is toward the 3′ end of the coding strand. Some genes on the same DNA molecule may be transcribed in opposite directions, so the upstream and downstream areas of the molecule may change depending on which gene is used as the reference.

“Host cell” includes any cell of any organism that is used for the purpose of producing a recombinant protein encoded by an expression vector or propagating the expression vector introduced into the host cell. A “mammalian recombinant host cell” refers to a mammalian host cell that comprises a heterologous expression vector, which may or may not be integrated into the host cell chromosome. A “bacterial recombinant host cell” refers to a bacterial host cell that comprises a heterologous expression vector, which may or may not be integrated into the host cell chromosome.

“Monoclonal antibody” or “mAb,” as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules constituting the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light chain” (about 25 kDa) and one “heavy chain” (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The variable regions of each light/heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.

III. Expression Vectors for Eukaryotic Expression System

In one aspect, provided herein are expression vectors designed for expressing recombinant proteins (e.g., biologics or vaccines) in eukaryotic cells. Recombinant proteins, such as monoclonal antibodies (mAb), are usually produced in mammalian host cells by stably integrating recombinant expression vectors encoding the recombinant protein into the host genome. In biologics development, stable cell line development is a long, complicated, and tedious process. Factors to be considered when designing expression vectors include but are not limited to: 1) efficient integration of the expression vector into the genomic transcriptionally active hot spots; 2) blockage of epigenetic gene silencing activities to ensure long term clone stability; 3) linkage of the GOI and the mammalian selection marker to ensure consistent expression of GOI in selected cells; 4) stable cells to withstand various processes, including continuous perfusion; and 5) fast selection timeline from construction to final clone selection.

In this disclosure, various DNA elements (e.g., regulatory elements, including but not limited to an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, or a UCOE) were selected and engineered into expression vectors. The effects of these engineered expression vectors on stable cell selection and expression level of recombinant proteins were evaluated. In certain embodiments, the innovative combinations of engineered DNA elements (e.g., regulatory elements, including but not limited to an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, or a UCOE) can generate high expression stable cells in a shorter timeline, compared to commercially available expression vectors.

For vector design, first, genome integration is a critical factor that directly impacts the efficiency and robustness in both stable cell line generation and long-term stability. Traditional methods of delivering the whole plasmid into the host cells is using random integration mechanisms through natural intracellular pathways, which lead to extremely low integration efficiency, such as 0.1-40% depending on the cell type and the delivery approach. In addition, the randomly selected integration sites are usually not optimal to support active expression, which reduces the probability of identifying high expressing cell lines. Further, the backbone of the expression vector typically contains elements that support plasmid replication in E. coli, which could cause epigenetic-mediated gene silencing after integration into the genome of mammalian cells. To solve these problems, a transposon technology is integrated herein. A transposon is a DNA sequence that can change its position within a genome. The class II transposons, also called DNA transposons, can efficiently transfer DNA via a “cut and paste” mechanism. A specific transposase recognizes transposon-specific ITR sequences located on both ends of the interested DNA fragment and efficiently moves the contents between the ITR sequences to the target chromosomal sites. It has been proposed that the preferred integration sites of these transposon systems are at the euchromatin regions, especially at AT-rich areas, which are potential genomic transcriptionally active hot spots. Another attractive feature of this transposon technology is that the transposase also enables the excision of the transposon in a completely seamless manner, leaving no sequences or mutations behind. Thus, through appropriate vector design, the transposase can remove the unfavorable bacteria-related element completely before integration into the mammalian genome to reduce epigenetic gene silencing. Furthermore, the transposon technology offers a large cargo-carrying capacity (up to 100-200 kb) which enables up to 10 times larger expression cassettes, compared to standard expression plasmid, to be easily integrated into target genome.

Second, the efficiency of delivering an expression vector into host cells is also very important. Depending on the cell type, vector size, DNA delivery approach, and DNA quality, the DNA delivery efficiency can range between 10% and 30%, which results in variations in selection timeline and quality of stably transfected host cells. Any common DNA delivery approach known in the art, such as biological approach (e.g., virus-mediated), chemical approach (e.g., cationic polymer, calcium phosphate, or cationic lipid), or physical approach (e.g., direct injection, biolistic particle delivery, electroporation, laser-irradiation, sonoporation, or magnetic nanoparticle) can be used to achieve optimal efficiency of delivering the expression vectors disclosed herein into host cells.

Third, how to effectively select stable high producers is important. One of the most reliable ways to achieve stable transfection is to select cells by applying a selective pressure, which can be overcome by stably incorporating the plasmid DNA containing an expression cassette encoding a drug-resistance enzyme (eukaryotic selectable marker) into the genome. There are multiple ways to design the expression cassette for the eukaryotic selectable marker. The eukaryotic selectable marker can be driven by a weaker promoter to increase selection stringency, which is one of the popular designs. In that design, the GOI is driven by a different promoter which is independent from the eukaryotic selection marker-containing cassette. Thus, the expression level of GOI in selected stable cells cannot be predicted or controlled, which can lead to low expression in transfection pools and/or clone instability. To resolve this issue, directly linking the GOI and the eukaryotic selection marker in the same expression cassette would be necessary. IRES is a type of regulatory element that can be found in several viruses and cellular RNAs (reviewed in McBratney et. al. Current Opinion in Cell Biology 5:961, 1993). It is an RNA element that allows for translation initiation in a cap-independent manner by recruiting ribosomes directly. Therefore, inserting an IRES sequence between two ORFs allows co-expression of the two genes together in a bicistronic eukaryotic expression cassette (Kaufman R. J., et al., Nucleic Acids Res 19:4485, 1991). The upstream gene translation is initiated at the normal 5′ cap, whereas the downstream gene translation is initiated at the IRES element, thereby resulting in co-expression of two independent proteins from a single mRNA transcript. Since IRES-mediated ribosome recruitment ratio is relatively lower, genes encoding drug-resistance enzymes are usually placed downstream of IRES, serving as selection markers. In addition, designing IRES sequence variants to reduce expression level of downstream selection markers can further increase the expression level of the upstream GOI, which is highly desirable in biological applications. However, modulating IRES strength by designing IRES sequence variants has its limitation and is often unpredictable because the effect also depends on other regulatory elements in the expression vector. In addition, manipulating IRES alone can cause cell stress, and sometimes cells cannot be recovered well as high producers. Thus, IRES has not been uniformly utilized in all mammalian stable transfection. In this disclosure, different IRES variants are evaluated and creatively combined with other regulatory elements in vector design to achieve high expression of the GOI and appropriate expression level of the eukaryotic selection marker for stable cell selection.

Fourth, transcription of eukaryotic genes is one of the key steps in protein expression, and it is regulated by a variety of cis- and trans-acting regulatory elements (reviewed by Dillon and Grosveld, Trends Genet. 9:134; 1993). Two of the best characterized cis regulatory elements are promoters and enhancers, which recruit RNA polymerase II and transcriptional activators. However, merely a promoter and an enhancer are not sufficient to consistently maintain a high expression of the GOI due to epigenetic inhibitory effects. Epigenetic effects are stably heritable phenotypes resulting from changes in a chromosome without alterations in the DNA sequence (Berger S L, et al. 2009. Genes & Development. 23:781). Among all epigenetics-mediated gene repression mechanisms, heterochromatinization and position-effect are common pathways that result in gene repression. Cis regulatory elements regulating the chromatin structure and prevent heterochromatinization include but are not limited to LCR (Grosveld F., et al., Cell 51:975, 1987), MAR (Phi-Van et al., Mol Cell Biol 10:2302; 1980), SAR (Gasser and Laemmli, Trends Genet 3:16, 1987), insulator (Kellum and Schedl, Cell 64:941, 1991), and EASE (Aldrich et al., Cytotechnology 28: 9, 1998). These elements have been shown to support relatively higher expression of linked genes at distal chromatin sites, although the complete mechanism is not fully understood. One of the common features of these cis elements is their AT-rich sequences, suggesting the lower propensity for chromosome condensation in the local region, which allows for efficient chromosomal transcription activation and prevents position-effect mediated gene silencing. Thus, adding these epigenetic/chromosome level regulatory elements into expression vectors can decrease epigenetic gene silencing and improve long-term stability of selected clones.

Some other cis regulatory elements, such as adenovirus TPL, can enhance protein expression at translation initiation and post-transcriptional levels (Kaufman R. J. PNAS (1985) 82:689). TPL comprises three introns, which are critical to the translation of adenovirus late mRNA in a cap-independent manner. In addition, this element has been suggested in the regulation of mRNA stability and mRNA nuclear export, which also impacts protein expression level. Thus, inserting the TPL sequence downstream of the promoter can increase the efficiency of gene expression significantly at post-transcriptional levels in certain cell types with selected promoters, especially for long mRNA transcripts.

Thus, in this disclosure, different combinations of various DNA elements (e.g., regulatory elements) are evaluated, and some combinations surprisingly decrease the timeline for selecting stable cells and generate high level of protein expression (e.g., up to 5-10 fold increase compared to commercially available expression vectors) in mammalian cells. The results are independent of host cell lines and consistent for various protein modalities, including monoclonal antibodies and Fc-fusion proteins.

In one aspect, provided herein is an expression vector comprising:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a promoter operably linked to an insertion site for a GOI, an IRES, a polynucleotide encoding a eukaryotic selectable marker, and a polyA signal;

(b) a second expression cassette comprising a polynucleotide encoding a bacterial selectable marker; and

The two expression cassettes may be arranged in the vector in any direction relative to each other. In some embodiments, transcriptions of the first and the second expression cassettes are in the same direction. In other embodiments, transcriptions of the first and the second expression cassettes are in the opposite directions.

The insertion site typically comprises at least one restriction enzyme (RE) recognition sequence, and may include two or more RE sequences to form a multiple cloning site.

In certain embodiments of the various expression vectors provided herein, the first expression cassette further comprises one or more regulatory elements. In some embodiments, the regulatory element is an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, or a UCOE. In one embodiment, the regulatory element is an enhancer. In another embodiment, the regulatory element is an insulator. In yet another embodiment, the regulatory element is a LCR. In still another embodiment, the regulatory element is a MAR. In one embodiment, the regulatory element is a SAR. In another embodiment, the regulatory element is an EASE. In yet another embodiment, the regulatory element is a TPL. In still another embodiment, the regulatory element is a UCOE. In some embodiments, the first expression cassette further comprises one regulatory element. In other embodiments, the first expression cassette further comprises two regulatory elements. In yet other embodiments, the first expression cassette further comprises three regulatory elements. In still other embodiments, the first expression cassette further comprises four regulatory elements. In some embodiments, the first expression cassette further comprises five regulatory elements. In other embodiments, the first expression cassette further comprises six regulatory elements. In yet other embodiments, the first expression cassette further comprises seven regulatory elements. In still other embodiments, the first expression cassette further comprises eight or more regulatory elements.

In some embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette. In other embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises one or more regulatory elements. In yet other embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises one or more regulatory elements selected from the group consisting of an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, and a UCOE. In certain embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises one regulatory element selected from the group consisting of an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, and a UCOE. In some embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises two regulatory elements selected from the group consisting of an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, and a UCOE. In other embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises three regulatory elements selected from the group consisting of an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, and a UCOE. In yet other embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises four regulatory elements selected from the group consisting of an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, and a UCOE. In still other embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises five regulatory elements selected from the group consisting of an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, and a UCOE. In some embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises six regulatory elements selected from the group consisting of an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, and a UCOE. In other embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises seven regulatory elements selected from the group consisting of an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, and a UCOE. In yet other embodiments, the expression vector further comprises two ITR sequences flanking the first expression cassette, and the first expression cassette further comprises eight regulatory elements selected from the group consisting of an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, a TPL, and a UCOE.

IRES that can be used in various embodiments of this disclosure include a wild type IRES from any species or a variant or mutant thereof, whether naturally occurred or man-made. Non-limiting examples of IRES that can be used include the following and variants thereof: the nucleotide sequence of the 5′ nontranslated region of encephalomyocarditis virus (EMCV) (GenBank: M81861.1; Duke et al., Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. J Virol. 1992 March; 66(3):1602-9.), IRES element described by Bochkov & Palmenberg (Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques. 2006 September; 41(3):283-4), IRES element from expression vector pInSRT-GFP (GenBank LC417349.1), IRES element from expression vector pCeMM-CTAP(SG) (GenBank EF467048.1), IRES element described by Jang & Wimmer (Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 September; 4(9):1560-72), IRES element from expression vector pIRESneo3 (Clontech/Takara Bio), IRES elements described in WO 2015/016786, WO 2015/021077, WO 2016/003368, WO 2016/074016, or WO 2013/092743, or variants thereof.

In other embodiments of the various expression vectors provided herein, the IRES comprises a polynucleotide sequence of SEQ ID NO:1, 2, 3, 23, 24, or 25. In one embodiment, the IRES comprises a polynucleotide sequence of SEQ ID NO:1. In another embodiment, the IRES comprises a polynucleotide sequence of SEQ ID NO:2. In yet another embodiment, the IRES comprises a polynucleotide sequence of SEQ ID NO:3. In one embodiment, the IRES comprises a polynucleotide sequence of SEQ ID NO:23. In another embodiment, the IRES comprises a polynucleotide sequence of SEQ ID NO:24. In yet another embodiment, the IRES comprises a polynucleotide sequence of SEQ ID NO:25. In one embodiment, the IRES comprises a polynucleotide sequence that is about 90, 95, 96, 97, 98, or 99% identical to the polynucleotide sequence of SEQ ID NO:1. In another embodiment, the IRES comprises a polynucleotide sequence that is about 90, 95, 96, 97, 98, or 99% identical to the polynucleotide sequence of SEQ ID NO:2. In yet another embodiment, the IRES comprises a polynucleotide sequence that is about 90, 95, 96, 97, 98, or 99% identical to the polynucleotide sequence of SEQ ID NO:3. In one embodiment, the IRES comprises a polynucleotide sequence that is about 90, 95, 96, 97, 98, or 99% identical to the polynucleotide sequence of SEQ ID NO:23. In another embodiment, the IRES comprises a polynucleotide sequence that is about 90, 95, 96, 97, 98, or 99% identical to the polynucleotide sequence of SEQ ID NO:24. In yet another embodiment, the IRES comprises a polynucleotide sequence that is about 90, 95, 96, 97, 98, or 99% identical to the polynucleotide sequence of SEQ ID NO:25.

In certain embodiments of the various expression vectors provided herein, the promoter is a human CMV immediate-early promoter, a human elongation factor 1 alpha (EF1a) promoter, a SV40 promoter, a phosphoglycerate kinase 1 (PGK1) promoter, a human ubiquitin C (Ubc) promoter, a human β-actin promoter, a CAG promoter, a yeast transcription elongation factor 1 (TEF1) promoter, a yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or a yeast alcohol dehydrogenase 1 (ADH1) promoter. In one embodiment, the promoter is a human CMV immediate-early promoter. In another embodiment, the promoter is a human EFla promoter. In yet another embodiment, the promoter is a SV40 promoter. In still another embodiment, the promoter is a PGK1 promoter. In one embodiment, the promoter is a human Ubc promoter. In another embodiment, the promoter is a human β-actin promoter. In yet another embodiment, the promoter is a CAG promoter. In still another embodiment, the promoter is a yeast TEF1 promoter. In one embodiment, the promoter is a yeast GAPDH promoter. In another embodiment, the promoter is a yeast ADH1 promoter.

In other embodiments of the various expression vectors provided herein, the insulator is HMR tRNA^Thr, Chal UAS, UASrpg, STAR, scs, scs′, gypsy, Fab-7, Fab-8, faswb, sns, UR1, RO, Lys 5′ A, HS4, 3′HS, BEAD-1, HS2-6, DMD/ICR, 5′HS5, apoB (−57 kb), apoB (+43 kb), or DM1. In one embodiment, the insulator is HMR tRNA^Thr. In another embodiment, the insulator is Chal UAS. In yet another embodiment, the insulator is UASrpg. In still another embodiment, the insulator is STAR. In one embodiment, the insulator is scs. In another embodiment, the insulator is scs′. In yet another embodiment, the insulator is gypsy. In still another embodiment, the insulator is Fab-7. In one embodiment, the insulator is Fab-8. In another embodiment, the insulator is faswb. In yet another embodiment, the insulator is sns. In still another embodiment, the insulator is UR1. In one embodiment, the insulator is RO. In another embodiment, the insulator is Lys 5′ A. In yet another embodiment, the insulator is HS4. In still another embodiment, the insulator is 3′HS. In one embodiment, the insulator is BEAD-1. In another embodiment, the insulator is HS2-6. In yet another embodiment, the insulator is DMD/ICR. In still another embodiment, the insulator is 5′HS5. In one embodiment, the insulator is apoB (−57 kb). In another embodiment, the insulator is apoB (+43 kb). In yet another embodiment, the insulator is DM1.

In yet other embodiments of the various expression vectors provided herein, the ITR is Tc1 ITR, Tc3 ITR, Minos ITR, Mos1 ITR, Famar1 ITR, Osmar5 ITR, Fot1 ITR, Impala ITR, ISY100 ITR, Mboumar-9 ITR, Sleeping Beauty ITR, Himar1 ITR, Frog Prince ITR, Hsmar1 ITR, SB100X ITR, piggyBac ITR, or Tol2 ITR. In one embodiment, the ITR is Tc1 ITR. In another embodiment, the ITR is Tc3 ITR. In yet another embodiment, the ITR is Minos ITR. In still another embodiment, the ITR is Mos1 ITR. In one embodiment, the ITR is Famar1 ITR. In another embodiment, the ITR is Osmar5 ITR. In yet another embodiment, the ITR is Fot1 ITR. In still another embodiment, the ITR is Impala ITR. In one embodiment, the ITR is ISY100 ITR. In another embodiment, the ITR is Mboumar-9 ITR. In yet another embodiment, the ITR is Sleeping Beauty ITR. In still another embodiment, the ITR is Himar1 ITR. In one embodiment, the ITR is Frog Prince ITR. In another embodiment, the ITR is Hsmar1 ITR. In yet another embodiment, the ITR is SB100X ITR. In still another embodiment, the ITR is piggyBac ITR. In one embodiment, the ITR is Tol2 ITR.

In still other embodiments of the various expression vectors provided herein, the polyA signal is a thymidine kinase gene polyA signal, a SV40 early gene polyA signal, a SV40 late gene polyA signal, a β-globin gene polyA signal, or the like. In some embodiments, the polyA signal is a thymidine kinase gene polyA signal. In certain embodiments, the polyA signal is a SV40 early gene polyA signal. In other embodiments, the polyA signal is a SV40 late gene polyA signal. In yet other embodiments, the polyA signal is a β-globin gene polyA signal.

A bacterial plasmid origin of replication is also present in various expression vectors disclosed herein to facilitate preparation of large quantities of the vector in bacteria cells. Non-limiting examples of plasmid replication origins include pUC origins derived from pBR322.

In another aspect, provided herein is an expression vector comprising:

(b) two ITR sequences flanking the first expression cassette;

(d) a bacterial plasmid origin of replication.

The insertion site typically comprises at least one RE recognition sequence, and may include two or more RE sequences to form a multiple cloning site.

In yet other embodiments of the various expression vectors provided herein, the ITR is Tc1 ITR, Tc3 ITR, Minos ITR, Mos1 ITR, Famar1 ITR, Osmar5 ITR, Fot1 ITR, Impala ITR, ISY100 ITR, Mboumar-9 ITR, Sleeping Beauty ITR, Himar1 ITR, Frog Prince ITR, Hsmar1 ITR, SB100X ITR, piggyBac ITR, or Tol2 ITR. In one embodiment, the ITR is Tc1 ITR. In another embodiment, the ITR is Tc3 ITR. In yet another embodiment, the ITR is Minos ITR. In still another embodiment, the ITR is Mos1 ITR. In one embodiment, the ITR is Famar1 ITR. In another embodiment, the ITR is Osmar5 ITR. In yet another embodiment, the ITR is Fot1 ITR. In still another embodiment, the ITR is Impala ITR. In one embodiment, the ITR is ISY100 ITR. In another embodiment, the ITR is Mboumar-9 ITR. In yet another embodiment, the ITR is Sleeping Beauty ITR. In still another embodiment, the ITR is Himar1 ITR. In one embodiment, the ITR is Frog Prince ITR. In another embodiment, the ITR is Hsmar1 ITR. In yet another embodiment, the ITR is SB100X ITR. In still another embodiment, the ITR is piggyBac ITR. In one embodiment, the ITR is Tol2 ITR.

Thus, in one particular embodiment, the expression vector comprises:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first HS4 insulator, an EASE, a human CMV immediate-early enhancer/promoter, a TPL, an insertion site for a GOI, an IRES comprising a polynucleotide sequence of SEQ ID NO:1, a polynucleotide encoding a glutamine synthetase, a polyA signal, and a second HS4 insulator;

(b) two piggyBac ITR sequences flanking the first expression cassette;

(d) a bacterial plasmid origin of replication.

In one particular embodiment, the expression vector comprises:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first HS4 insulator, an EASE, a human CMV immediate-early enhancer/promoter, a TPL, an insertion site for a GOI, an IRES comprising a polynucleotide sequence of SEQ ID NO:2, a polynucleotide encoding a glutamine synthetase, a polyA signal, and a second HS4 insulator;

(b) two piggyBac ITR sequences flanking the first expression cassette;

(d) a bacterial plasmid origin of replication.

In one particular embodiment, the expression vector comprises:

(b) two piggyBac ITR sequences flanking the first expression cassette;

(d) a bacterial plasmid origin of replication.

In one particular embodiment, the expression vector comprises:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first HS4 insulator, an EASE, a human CMV immediate-early enhancer/promoter, a TPL, an insertion site for a GOI, an IRES comprising a polynucleotide sequence of SEQ ID NO:23, a polynucleotide encoding a glutamine synthetase, a polyA signal, and a second HS4 insulator;

(b) two piggyBac ITR sequences flanking the first expression cassette;

(d) a bacterial plasmid origin of replication.

In one particular embodiment, the expression vector comprises:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first HS4 insulator, an EASE, a human CMV immediate-early enhancer/promoter, a TPL, an insertion site for a GOI, an IRES comprising a polynucleotide sequence of SEQ ID NO:24, a polynucleotide encoding a glutamine synthetase, a polyA signal, and a second HS4 insulator;

(b) two piggyBac ITR sequences flanking the first expression cassette;

(d) a bacterial plasmid origin of replication.

In one particular embodiment, the expression vector comprises:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first HS4 insulator, an EASE, a human CMV immediate-early enhancer/promoter, a TPL, an insertion site for a GOI, an IRES comprising a polynucleotide sequence of SEQ ID NO:25, a polynucleotide encoding a glutamine synthetase, a polyA signal, and a second HS4 insulator;

(b) two piggyBac ITR sequences flanking the first expression cassette;

(d) a bacterial plasmid origin of replication.

In one specific embodiment, the EASE comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:12. In another embodiment, the EASE comprises the polynucleotide sequence of SEQ ID NO:12. In yet another embodiment, the EASE consists of the polynucleotide sequence of SEQ ID NO:12.

In one specific embodiment, the TPL comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:17. In another embodiment, the TPL comprises the polynucleotide sequence of SEQ ID NO:17. In yet another embodiment, the TPL consists of the polynucleotide sequence of SEQ ID NO:17.

In one specific embodiment, the gene encoding the glutamine synthetase comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:21. In another embodiment, the gene encoding the glutamine synthetase comprises the polynucleotide sequence of SEQ ID NO:21. In yet another embodiment, the gene encoding the glutamine synthetase consists of the polynucleotide sequence of SEQ ID NO:21.

In one specific embodiment, the human CMV promoter comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:18. In another embodiment, the human CMV promoter comprises the polynucleotide sequence of SEQ ID NO:18. In yet another embodiment, the human CMV promoter consists of the polynucleotide sequence of SEQ ID NO:18.

In one specific embodiment, the SV40 promoter comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:15. In another embodiment, the SV40 promoter comprises the polynucleotide sequence of SEQ ID NO:15. In yet another embodiment, the SV40 promoter consists of the polynucleotide sequence of SEQ ID NO:15.

In one specific embodiment, the SV40 enhancer comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:22. In another embodiment, the SV40 enhancer promoter comprises the polynucleotide sequence of SEQ ID NO:22. In yet another embodiment, the SV40 enhancer promoter consists of the polynucleotide sequence of SEQ ID NO:22.

In one specific embodiment, the human CMV immediate-early enhancer/promoter comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:16. In another embodiment, the human CMV immediate-early enhancer/promoter comprises the polynucleotide sequence of SEQ ID NO:16. In yet another embodiment, the human CMV immediate-early enhancer/promoter consists of the polynucleotide sequence of SEQ ID NO:16.

In one specific embodiment, the HS4 insulator comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:11. In another embodiment, the HS4 insulator comprises the polynucleotide sequence of SEQ ID NO:11. In yet another embodiment, the HS4 insulator consists of the polynucleotide sequence of SEQ ID NO:11.

In one specific embodiment, the 5′ piggyBac ITR comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:9, and the 3′ piggyBac ITR comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:10. In another embodiment, the 5′ piggyBac ITR comprises the polynucleotide sequence of SEQ ID NO:9, and the 3′ piggyBac ITR comprises the polynucleotide sequence of SEQ ID NO:10. In yet another embodiment, the 5′ piggyBac ITR consists of the polynucleotide sequence of SEQ ID NO:9, and the 3′ piggyBac ITR consists of the polynucleotide sequence of SEQ ID NO:10.

In one specific embodiment, the β-globin gene polyA signal comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:13. In another embodiment, the β-globin gene polyA signal comprises the polynucleotide sequence of SEQ ID NO:13. In yet another embodiment, the β-globin gene polyA signal consists of the polynucleotide sequence of SEQ ID NO:13.

In one specific embodiment, the SV40 late gene polyA signal comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:14. In another embodiment, the SV40 late gene polyA signal comprises the polynucleotide sequence of SEQ ID NO:14. In yet another embodiment, the SV40 late gene polyA signal consists of the polynucleotide sequence of SEQ ID NO:14.

In one specific embodiment, the ampicillin resistance gene comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:20. In another embodiment, the ampicillin resistance gene comprises the polynucleotide sequence of SEQ ID NO:20. In yet another embodiment, the ampicillin resistance gene consists of the polynucleotide sequence of SEQ ID NO:20.

In one specific embodiment, the bacterial plasmid origin of replication comprises a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:19. In another embodiment, the bacterial plasmid origin of replication comprises the polynucleotide sequence of SEQ ID NO:19. In yet another embodiment, the bacterial plasmid origin of replication consists of the polynucleotide sequence of SEQ ID NO:19.

In one particular embodiment, the expression vector comprises:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first HS4 insulator comprising a polynucleotide sequence of SEQ ID NO:11, an EASE comprising a polynucleotide sequence of SEQ ID NO:12, a human CMV immediate-early enhancer/promoter comprising a polynucleotide sequence of SEQ ID NO:16, a TPL comprising a polynucleotide sequence of SEQ ID NO:17, an insertion site for a GOI, an IRES comprising a polynucleotide sequence of SEQ ID NO:3, a glutamine synthetase gene comprising a polynucleotide sequence of SEQ ID NO:21, a polyA signal comprising a polynucleotide sequence of SEQ ID NO:13, and a second HS4 insulator that is identical to the first HS4 insulator but in the opposite direction;

(b) two piggyBac ITR sequences flanking the first expression cassette, wherein the 5′ piggyBac ITR comprises a polynucleotide sequence of SEQ ID NO:9, and wherein the 3′ piggyBac ITR comprises a polynucleotide sequence of SEQ ID NO:10;

(c) a second expression cassette comprising a polynucleotide encoding a bacterial selectable marker, wherein the polynucleotide encoding the bacterial selectable marker comprises a polynucleotide sequence of SEQ ID NO:20; and

(d) a bacterial plasmid origin of replication, wherein the bacterial plasmid origin of replication comprises a polynucleotide sequence of SEQ ID NO:19.

In another particular embodiment, the expression vector comprises:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first HS4 insulator consisting of a polynucleotide sequence of SEQ ID NO:11, an EASE consisting of a polynucleotide sequence of SEQ ID NO:12, a human CMV immediate-early enhancer/promoter consisting of a polynucleotide sequence of SEQ ID NO:16, a TPL consisting of a polynucleotide sequence of SEQ ID NO:17, an insertion site for a GOI, an IRES consisting of a polynucleotide sequence of SEQ ID NO:3, a glutamine synthetase gene consisting of a polynucleotide sequence of SEQ ID NO:21, a polyA signal consisting of a polynucleotide sequence of SEQ ID NO:13, and a second HS4 insulator that is identical to the first HS4 insulator but in the opposite direction;

(b) two piggyBac ITR sequences flanking the first expression cassette, wherein the 5′ piggyBac ITR consists of a polynucleotide sequence of SEQ ID NO:9, and wherein the 3′ piggyBac ITR consists a polynucleotide sequence of SEQ ID NO:10;

(c) a second expression cassette comprising a polynucleotide encoding a bacterial selectable marker, wherein the polynucleotide encoding the bacterial selectable marker consists of a polynucleotide sequence of SEQ ID NO:20; and

(d) a bacterial plasmid origin of replication, wherein the bacterial plasmid origin of replication consists of a polynucleotide sequence of SEQ ID NO:19.

In another specific embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 26, 27, 28, or 29. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:4. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:5. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:6. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:7. In yet still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:8. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:26. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:27. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:28. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence of SEQ ID NO:29.

In another specific embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 26, 27, 28, or 29. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:4. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:5. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:6. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:7. In yet still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:8. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:26. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:27. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:28. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 85% identical to the polynucleotide sequence of SEQ ID NO:29.

In another specific embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 26, 27, 28, or 29. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:4. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:5. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:6. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:7. In yet still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:8. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:26. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:27. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:28. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO:29.

In another specific embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 26, 27, 28, or 29. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:4. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:5. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:6. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:7. In yet still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:8. In one embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:26. In another embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:27. In yet another embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:28. In still another embodiment, the expression vector comprises a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO:29.

Polypeptides that can be encoded by the GOI and expressed by various expression vectors described herein include, but are not limited to, therapeutic or prophylactic polypeptides such as adhesion molecules, antibody light and/or heavy chains, cytokines, enzymes, lymphokines, and receptors, etc.

In yet another aspect, provided herein is a mammalian recombinant host cell comprising a mammalian host cell transfected with the expression vector described herein.

Suitable mammalian host cells include but are not limited to hamster cells, such as CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1, CHO-DG44, CHO-DBX11, CHOK1SV™, HD-BIOP1, CHOZN®, BHK21, BHK TK⁻, or ExpiCHO, as well as derivatives/descendants of these hamster cell lines. Also suitable are myeloma cells from the mouse, such as NSO or Sp2/0-AG14 cells, and human cell lines, such as HEK293, Hela, Jerkat, TP1, or PER.C6, as well as derivatives/descendants of these mouse and human cell lines.

In certain embodiments of various mammalian recombinant host cells provided herein, the mammalian host cell is a CHO cell. In one embodiment, the endogenous glutamine synthetase gene of the CHO cell is knocked out. In another embodiment, the mammalian host cell is a CHOK1SV™ cell. In yet another embodiment, the mammalian host cell is a HD-BIOP1 cell. In still another embodiment, the mammalian host cell is a CHOZN® cell.

In another aspect, provided herein is a bacterial recombinant host cell comprising a bacterial host cell transformed with the expression vector described herein.

Suitable bacterial host cells include but are not limited to the bacterial host cells that are commonly used for molecular cloning, transformation, and/or propagation of expression vectors by an ordinary person in the art, for example, DH5α™, DH10B™, JM109, TOP10, etc., as well as derivatives and modifications of them.

In certain embodiments, provided herein is a method of propagating an expression vector, comprising culturing the bacterial recombinant host cell described herein, under conditions in which the expression vector is replicated, and recovering the expression vector from the culture.

In still another aspect, provided herein is a mammalian recombinant host cell comprising a mammalian host cell co-transfected with a first expression vector and a second expression vector, wherein the first expression vector and the second expression vector are the expression vector described herein, wherein the GOI of the first expression vector encodes a light chain of a monoclonal antibody and the GOI of the second expression vector encodes a heavy chain of the monoclonal antibody, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In one embodiment, provided herein is a CHO recombinant host cell comprising a CHO host cell co-transfected with a first expression vector and a second expression vector, wherein the first expression vector and the second expression vector are the expression vector described herein, wherein the GOI of the first expression vector encodes a light chain of a monoclonal antibody and the GOI of the second expression vector encodes a heavy chain of the monoclonal antibody, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In another embodiment, provided herein is a CHO recombinant host cell comprising a CHO host cell co-transfected with a first expression vector and a second expression vector, wherein the endogenous glutamine synthetase gene of the CHO cell is knocked out, wherein the first expression vector and the second expression vector are the expression vector described herein, wherein the GOI of the first expression vector encodes a light chain of a monoclonal antibody and the GOI of the second expression vector encodes a heavy chain of the monoclonal antibody, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In yet still another aspect, provided herein is a method of producing a monoclonal antibody, comprising culturing the mammalian recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed.

In some embodiments, provided herein is a method of producing a monoclonal antibody, comprising culturing the mammalian recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and recovering the monoclonal antibody from the culture.

In certain embodiments, the method of producing a monoclonal antibody comprises culturing the mammalian recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In other embodiments, the method of producing a monoclonal antibody comprises culturing the mammalian recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and recovering the monoclonal antibody from the culture, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In one embodiment, provided herein is a method of producing a monoclonal antibody, comprising culturing the CHO recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed.

In another embodiment, provided herein is a method of producing a monoclonal antibody, comprising culturing the CHO recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and recovering the monoclonal antibody from the culture.

In certain embodiments, the method of producing a monoclonal antibody comprises culturing the CHO recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In other embodiments, the method of producing a monoclonal antibody comprises culturing the CHO recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and recovering the monoclonal antibody from the culture, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In another embodiment, provided herein is a method of producing a monoclonal antibody, comprising culturing the CHO recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, wherein the endogenous glutamine synthetase gene of the CHO cell is knocked out.

In certain embodiments, the method of producing a monoclonal antibody comprises culturing the CHO recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, wherein the endogenous glutamine synthetase gene of the CHO cell is knocked out, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In certain embodiments, the method of producing a monoclonal antibody comprises culturing the CHO recombinant host cell co-transfected with a first expression vector comprising a first GOI encoding the light chain of the monoclonal antibody and a second expression vector comprising a second GOI encoding the heavy chain of the monoclonal antibody described herein, under conditions in which both the light chain and the heavy chain of the monoclonal antibody are expressed, and recovering the monoclonal antibody from the culture, wherein the endogenous glutamine synthetase gene of the CHO cell is knocked out, and wherein the eukaryotic selectable marker of the first expression vector is different from the eukaryotic selectable marker of the second expression vector.

In any embodiments of various expression vectors, host cells, methods of propagating expression vectors, methods of producing polypeptides encoded by the GOI, or methods of producing monoclonal antibodies, the polynucleotide sequence for an individual vector element or component (e.g., regulatory elements, including but not limited to an enhancer, an insulator, a LCR, a MAR, a SAR, an EASE, or a TPL) can be obtained from a different species than the species from which the sequences disclosed herein are obtained. For example, a species variant of a human β-globin polyA signal, such as a mouse or hamster β-globin polyA signal, can be used in the expression vectors. Similarly, a species variant of an adenovirus TPL, such as a human adenovirus B TPL, a human adenovirus C TPL, a human adenovirus E TPL, or an ovine adenovirus TPL, can be used in the expression vectors.

EXAMPLES

These examples are intended to further clarify the present invention and not to limit the invention. Any composition or method, in whole or in part, set forth in the examples form a part of the present invention.

Example 1: Construction of Expression Vectors

FIG. 1 illustrates an exemplary innovative expression vector comprising a combination of engineered elements, which were selected to maximize protein expression in mammalian cells. In FIG. 1, E represents an enhancer; P represents a promoter; ORF represents an open reading frame; UTR represents an untranslated region; IRES represents an internal ribosome entry site; PolyA represents a polyadenylation signal; ITR represents an inverted repeat region, Bac-Ori represents a bacterial origin of replication; Amp represents an ampicillin selectable marker.

The DNA sequence information of plasmid pUC19 and various fragments, such as EASE, CMV Enhancer/Promoter, TPL, IRES, mammalian selection marker, and PolyA, were obtained from the public domain. The polynucleotides of pUC19 and these fragments were synthesized by Blue Heron Biotech, LLC, WA. A variety of expression vectors with GOI were constructed as shown in FIGS. 2A-2E, using NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Beverly, Mass.).

The DNA sequences of selected elements or expression vectors are shown as follows:

IRES-1 (Genes Dev. 4(9):1560-72 (1990), SEQ ID NO: 1):

cccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgaggg

cccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaag

cagttectctggaagatcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcgg

ccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctc

tcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgt

gtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaacc

IRES-2 (SEQ ID NO: 2):

cccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgaggg

cccggaaacctggccctgtatcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaag

cagttectctggaagatcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcgg

ccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctc

tcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgt

gtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa

IRES-3 (SEQ ID NO: 3):

cccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgaggg

cccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaag

cagttectctggaagatcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcgg

ccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctc

tcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgt

gtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacg

IRES-4 (SEQ ID NO: 23):

cccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgaggg

cccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaag

cagttectctggaagatcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcgg

ccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctc

tcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgt

gtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaacc

IRES-5 (SEQ ID NO: 24):

cccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgaggg

cccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaag

cagttectctggaagatcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcgg

ccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctc

tcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgt

gtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa

IRES-6 (SEQ ID NO: 25):

cccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgaggg

cccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaag

cagttectctggaagatcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcgg

ccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctc

tcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgt

gtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacg

pCLD116 (SEQ ID NO: 4):

1
CCAATGATCT TAAGTTAACC CTAGAAAGAT AATCATATTG TGACGTACGT TAAAGATAAT CATGCGTAAA ATTGACGCAT

81
GTGTTTTATC GGTCTGTATA TCGAGGTTTA TTTATTAATT TGAATAGATA TTAAGTTTTA TTATATTTAC ACTTACATAC

161
TAATAATAAA TTCAACAAAC AATTTATTTA TGTTTATTTA TTTATTAAAA AAAAACAAAA ACTCAAAATT TCTTCTATAA

241
AGTAACAAAA CTTTTATCGA ATTTGCAGCC CGGGACTAGC TAGAGGGACA GCCCCCCCCC AAAGCCCCCA GGGATGTAAT

321
TACGTCCCTC CCCCGCTAGG GGGCAGCAGC GAGCCGCCCG GGGCTCCGCT CCGGTCCGGC GCTCCCCCCG CATCCCCGAG

401
CCGGCAGCGT GCGGGGACAG CCCGGGCACG GGGAAGGTGG CACGGGATCG CTTTCCTCTG AACGCTTCTC GCTGCTCTTT

481
GAGCCTGCAG ACACCTGGGG GGATACGGGG AAAACTTAAG ATCCGACCGG TGCTGTGGAA TGTGTGTCAG TTAGGGTGTG

561
GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC

641
CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC

721
CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG CTCCATCGTT CAGATTTTAC CACATTTGTA GAGGTTTTAC

801
TTGCTTTAAA AAACCTCCCA CACCTCCCCC TGAACCTGAA ACATAAAATG AATGCAATTG TTGTTGTTAA CTTGTTTATT

881
GCAGCTTATA ATGGTTACAA ATAAAGCAAT AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG

961
TGGTTTGTCC AAACTCATCA ATGTATCTTA TCATGTCTGC TCGAAGCGGC CGGCCGCCCC GACTCTAGAT TAGTTTTTGT

1041
ATTGGAAGGG CTCGTCGCCA GTCTCATTGA GAAGGCATGT GCGGACGATG GCTTCTGTCA CTGCAAAGGG GTCACAATTG

1121
GCAGAGGGGC GGCGGTCTTC AAAGTAACCT TTCTTCTCCT GGCCGACAGT CCGGGGAATG CGGATGCTGG CACTGCGATT

1201
GGCGACACCA GCAGAAAAGT CGTTGATGTT GGACGTTTCG TGGAACCCAG TCAGACGACG GGCATTGTCC AGGCCCCCCT

1281
TGGGATCGTA GGCTCGAATG TGGTACCGGT GCCGCTTGCT TAGTTTCTCG ATGGCCTCCT CGATGTGCTT CAGACCATTC

1361
TCCTCCCGCA TGGCCTTGGT GCTAAAGTTG GTATGGCAGC CTGCACCATT CCAGTTCCCA GGAATGGGCT TGGGGTCAAA

1441
GGTTGCTATT ACCCCAAAGT CTTCACATAC TCGATGCAAG ATGAAACGGG CCACCCAGAG ATGATCTCCC ATGCGGATTC

1521
CTTCACAGGG TCCTATTTGG AACTCCCACT GGGCAGGCAT GACCTCAGCA TTTGTTCCTG TAATCTTGAC CCCAGCATAC

1601
AAGCAGGCGC GGTAGTGAGC CTCCACGATA TCCCTGCCAT AGGCTTTGTC TGCGCCCACA CCACAGTAAT ACGGACCTTG

1681
GGGCCCAGGA AAGCCATTGG AAGGCCAACC AAAAGGGTGC CCATCTGTTC CCATCAGAGT ATACTCCTGT TCCATTCCAA

1761
ACCAGGGGTG CTGGTTGCTC ACCATGTCCA TTATCCGTTT ACACGAGTGC CTTAAATTGG TCTCTGCAGG CTTCCGGTTG

1841
TACTTGAAAA CTTCACAGAA CACCAGCTTG TTGGGATCTC TGCGGAAGGG GTCCCGAAAC ATGGCAACAG GGCTGAGATA

1921
CATGTCACTG TTGGAGCCCT CAGACTGAAA GGTACTAGAG CCATCAAAAT TCCACTCAGG TAACTCTTCT ACACACTTGG

2001
GCTCACAGTC CAGGGTGCGG GTTTTGCAGC GCAGTCCTTC TCCAGTACCA TCAACCCAGA TATACATGGC TTGGACTTTC

2081
TCACCCTGGG GCAGGCACAA GTACATTTGC TTGATGTTTT TGTTCAAGTG GGAACTTGCT GAGGTGGCCA TGGTGGCGGC

2161
TTTGCAAAAG CCTAGGCCTC CAAAAAAGCC TCCTCACTAC TTCTGGAATA GCTCAGAGGC CGAGGCGGCC TCGGCCTCTG

2241
CATAAATAAA AAAAATTAGT CAGCGATGGG GCGGAGAATG GGCGGAACTG GGCGGAGTTA GGGGCGGGAT GGGCGGAGTT

2321
AGGGGCGGGA CTATGGTTGC TGACTAATTG AGATGCAATC ACTGACACAC ATTCCACAGC TGCCTCGCGC GTTTCGGTGA

2401
TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG GTCACAGCTT GTCTGTAAGC GGATGCCGGG AGCAGACAAG

2481
CCCGTCAGGG CGCGTCAGCG GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG CGGAGTGTAT

2561
ACTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACTGAG CCTCGAGCGA TGTACGGGCC AGATATACGC GTTGACATTG

2641
ATTATTGACT AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC

2721
TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA TGACGTATGT TCCCATAGTA

2801
ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGACT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA

2881
TCATATGCCA AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT

2961
GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC GGTTTTGGCA GTACATCAAT

3041
GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA

3121
AAATCAACGG GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA CGGTGGGAGG

3201
TCTATATAAG CAGAGCTCTC TGGCTAACTA GAGAACCCAC TGCTTACTGG CTTATCGAAA TTAATACGAC TCACTATAGC

3281
AATTGCACGT GTGGCCACAG GTAAGTTTAA AGCTCAGGTC GAGACCGGGC CTTTGTCCGG CGCTCCCTTG GAGCCTACCT

3361
AGACTCAGCC GGCTCTCCAC GCTTTGCCTG ACCCTGCTTG CTCAACTCTA CGTCTTTGTT TCGTTTTCTG TTCCTTTCTC

3441
TCCACAGGCG GATCCGAATT CTGAAGATCT AGATCCCCCT CGCTTTCTTG CTGTCCAATT TCTATTAAAG GTTCCTTTGT

3521
TCCCTAAGTC CAACTACTAA ACTGGGGGAT ATTATGAAGG GCCTTGAGCA TCTGGATTCT GCCTAATAAA AAACATTTAT

3601
TTTCATTGCA ATGATGTATT TAAATTATTT CTGAATATTT TACTAAAAAG GGAATGTGGG AGGTCAGTGC ATTTAAAACA

3681
TAAAGAAATG AAGAGGGGGA TCTTCGCGAA TCCATCGATG AGGGACAGCC CCCCCCCAAA GCCCCCAGGG ATGTAATTAC

3761
GTCCCTCCCC CGCTAGGGGG CAGCAGCGAG CCGCCCGGGG CTCCGCTCCG GTCCGGCGCT CCCCCCGCAT CCCCGAGCCG

3841
GCAGCGTGCG GGGACAGCCC GGGCACGGGG AAGGTGGCAC GGGATCGCTT TCCTCTGAAC GCTTCTCGCT GCTCTTTGAG

3921
CCTGCAGACA CCTGGGGGGA TACGGGGAAA ATAGACACCG CGGTGGAGCT CCAGCTTTTG TTCCCTTTAG TGAGGGTTAA

4001
TTAGTTCTTA ATACGACTCA CTATAGGGCG AATTGGCTAC CGGGCCGCCC ATCGAGGGTA TCATAAGCTT ATATCTATAA

4081
CAAGAAAATA TATATATAAT AAGTTATCAC GTAAGTAGAA CATGAAATAA CAATATAATT ATCGTATGAG TTAAATCTTA

4161
AAAGTCACGT AAAAGATAAT CATGCGTCAT TTTGACTCAC GCGGTCGTTA TAGTTCAAAA TCAGTGACAC TTACCGCATT

4241
GACAAGCACG CCTCACGGGA GCTCCAAGCG GCGACTGAGA TGTCCTAAAT GCACAGCGAC GGATTCGCGC TATTTAGAAA

4321
GAGAGAGCAA TATTTCAAGA ATGCATGCGT CAATTTTACG CAGACTATCT TTCTAGGGTT AAATCGATAT CGGAAAGAAC

4401
ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC

4481
TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC

4561
CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC

4641
GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA

4721
ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC

4801
CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT

4881
AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG

4961
CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

5041
GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC

5121
ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA

5201
GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

5281
TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA

5361
GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC

5441
TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

5521
TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA

5601
AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT

5681
GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

5761
TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG

5841
GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT

5921
CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

6001
TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA

6081
CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA

6161
AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTC

pCLD21 (SEQ ID NO: 5):

1
CCTTCCCTTC TACCACACCC TAATTGTAAT CCATTTTAAT TTCCTGGTCA CAGTCCTGTC TCTCCTTCCA TTGTACCTTG

81
CCCTTTTCTA AAGAGCGACT GCAAAGTATG TTTGCGTAGG TGAGGATCTA AAACTTTATG AGGTACGAAC ATCACAGAAT

161
TACTTTGTAA TTTCAGTTTA TTGTAGGCTT GGCTTTTTGG GGAGGGTTTA CGTCTTAGAC CTCTTAGTGC TTCTTTGTTT

241
CATGGTGTTC TAACTTCGAA GCATCTCTGT AGCTTTAATG GATTCCTTTT CTGAAAGCTT TGCTCTCTTT CTTCCCCCTC

321
GGCTTTCTCT TAGGCAAGAG GGCTAACTGT AAAGTAAGGC TTACTGCCTT GTGTTTCCAA ATGTGTCCGA AGAGGAAGTG

401
TCTTCTGTGA ATCCTGTTAT GCATGAATAA CAGGAAATAG AAAGAAATTC ACTTTCATTA TTATAAAAGT AATATGTTCG

481
TTTAAAAAAT TCTAATGAAG AGCTGGAGAT GCAACCCAGG GGTAGAGCAC ACACTCAGCA TGCAGGAGGC CCTGGGTCCA

561
ATCTTGGAAT CTCCTCTCAG TTAACCTGAT CTCTAGCTGA TTAGTAGTGA GTGCAAGCCC ACTTTCCTCT TCTGCCTCAT

641
TGCTCAGTGA TAACAGCTGT TAAACTTTGT CTTATTCTAA AACTACCTCT GTGCAAATGC TAGCACAATA ATATATATCA

721
TATGCACATG ATTTTTTTTT TATCTTGAAA AGTAAGTCAG TATAGCTACA AAGTTCACTT GGCATTGTCA ACATTTCACA

801
GGCGTAATAT TCCTCCTCTA GTACTGTCCT CTTCATTCTT TGTGACCAAG TTTGGAGAGA GTGCACAAAT GCCAGGGAGG

881
TTTGTGGGAA GGTTTCTCAT GTTCTGGTAA GGCGAGTAAG AAAATAGTCT CATGCAGGTG AAATGAGTGC TATGCAGTAT

961
ATATTATACC AGAGAACAGC AAATGACCAA ATTCACACTG AACTAGTTCA GTAAAATTGG CTTTGTCAAA GCTTTCCTTG

1041
CTTAAAATGT AATTCCCTGT CATCCTAGTT CTGGTCTGGA TTCTTTTCCT GGAGTCTTGA CTTCCAGATT CCCTGTGGAC

1121
TTTTGTTTGA GTTTCAAGCT TTTGAAATAT AGAAACCTAT CTAACTTAAC AAACTTGGGA GAGAAAAGAC TCCAGAACAA

1201
CTGAAAACAG ACCAGGCTAA ATGAATAGAC TTTATTCCTC TCTTCTTACC TGCAGTTTTC AGATATGCAG AGTTGGAGCG

1281
GATCTTAGAG GTTGATTCAT TCATGCCTGA AGAAAACACA TTTTATAGAC CCTGTGCCCA AGTTCGTGGT GGACATCACC

1361
CTTTATTTAC TAATTGCACT ACATAACAGG CATTTTAGAA GACTGCTCCA GTCAGAGACC CCGCCTTAGA GGAATCTGTA

1441
AACCCTGAAC TCCTATCACT CATGAGCACT AGTTATGTTT GGAATGCCGT ATTAAAACAA AAGTTACATT TCTAAACTTA

1521
AAATTTTCTA GCACAGAGAC AGTGGGAGTA GCTAACTTTG ATAGACATTT TTCTACTAAA AGTCTTTCTA AGTACATAAT

1601
CTTCTGTAAG TTGGAAAACA GCAAAATAGA ACGTCTCCTA CGTAGTTAAT CTTTTTGCAT AATTTGCACA TGTAGGAGTT

1681
ATTAGTATAC GGGTAAGTTT TCACTTTTTC CCCCAACTGG AGTGTCTTGT GGCTGGGTTT GAAAAAGGGA ACGGGAGGCC

1761
GCTGGAGGGG ATTGGTAAAT GAGATAAAAC ACCACTCATT CAACTCAGTG ACTCAGCATT TAAATTTTCC ATAAAAGGAT

1841
TAAAGGAAAA TTAAACAAAT TCTTAAAGCC AAGACTCTGG AGAAACTTGT TGGTGTGCTT TAGTTTTCAC TGTTATGACT

1921
CATGAATTTA TGCATAAATT AGTACATTTA TAAAAACATA GCCTTTTTAG AGTTTTCTGT TTGGCTAAAG TGCCATTGTT

2001
AGCATTTGGA ATTACCTTTT TATGTCTTAT ATTTTTTCCA AATAAAAATA AATGTTTCTG CTGTCTTACT ACTGAAACTA

2081
CGTTGTGAGC ACTTTAAATT TCTCAAAGCA GTTTCGCCTG TTATACTTGG CGCTTAGTCA TCGTCGTACA CAACAGGACC

2161
TGATTAAGAA GGCTGTGCTG CCTCTAAGCC GGGCTAGATT GTAGCCACTA GCAACCAGGC TGCAATAATT TCCCTTTGAT

2241
GACATCATCC ACTGTGGAAG AACCCAGTTG CTTCAGCCAG TCGAACTATA CAGTTCCAAC CTCATCAAAT ATGGCATCTC

2321
CCTTGCCTGC TATAGCAGGG GGAGGAAAAA ATGCCACCAT CTTTTTAATC TAGCAAGCTT CTCTTTTCTT CATCTTTTTT

2401
TTTTTCTTTT AAAAAAATTC TGATCATGGA TGCTTCTTCC GATCCCTATT TGCCTTATGA CGGGGGAGGA GACAATATCC

2481
CCTTGAGGGA ATTACATAAA AGAGGTAAGA GCATCCCCTT GCTCTGAATC CTCTGTTGGT TGTTGTGCAT GCGGCTGGGC

2561
GGTTCTGGGG ACAGGCTGTC TGTTGTCCTC TTGCTGCAAT GTGCTGCTTA GTTGCCCTGC CTTGTTGCTG TGGGAGAATG

2641
CGACCTTCCC AGCAGGGCTG GCCCTCCCTG ATTGTTTGCT CTGTGCAGAT TAGCCCTGCT TCAGATCACA TAGGGCTGCA

2721
GACTCCATCT TCTGTGTGAA AATGCTTTCG GTTTGATTGC AGAAATAAGC TGCCTTTACA GCCAGCTAAA GTCCTGGTGG

2801
TTGGTTGGCA CCTGCAAAGT AGTATTTTTG TACCTCTGGA AACTTATATT TTCTTTACAC AGCAATATCA AGTGCCGGTA

2881
TGCCATTCTG TTTTGGCTGC TGCCAATTAC CATGTAGACT TTGCACCACA GAGTAATAGT AAAAGCTCCT AGCTGCATTT

2961
TATAACATTT AAAAATAGCA GGAAAGAAGA ATTATTTTTG ATTTAACATG TTTTTGTCAT TTAACGTCTT AACTGATTGA

3041
CATACTATAT TGTCTGTCTC GTGGGTATCT TGTACAACTT GATAGGATAA AGCAATTTAG TTTTTTTTTT TTTTTTTAAA

3121
TACATCCAGA ATGTAAGTCG TCAGTAGTTT TCGAACAGAT AAGTAATGGT GTTAATCTTT TGGCAGGCTT TGCCTTGGTC

3201
TCCTTAAAGC TAATTAGGTG TTACTTAATT AAACTGCTCT TTTGCTCATT TTCTTAAATT ATTTTTTTAA AAGATAGTTG

3281
GCATTTGCTG TTCTAGAAAT AAACTTCAAG AAACATTCTT TAGCCAGATG ACTTCATGTA TGAGCCATGT TAGTTTGAAT

3361
TATTTGCTTG GTGTTATAAA CTTTATGGTT TAATACCAAC TTTTATTATG TTTACAAGGT AAATAAGGAA AATTTCAAGT

3441
ACATTTTGTA TCCTGAGAAC AAATTTAAGT TCCATAGAAT TTAGGAATTA CAATGTATTC AACAGATACT TACTTGTCAT

3521
ACTGTGCCTG CAAAACAATA ATTAGACTCT GAACAGGTGC AACAATTTTC TGTAGAATTA GACAAGTCTT CTTTTGGCAG

3601
GTGTTACTAA GTAGGCCATT TCCCAAGGAA CAGGGAATTT GCCAGGCTTT TGTGGTGGAG AGAATAGAAT GAATAAATGC

3681
TGTGGGGAGT AAAGAGCTTG TCAGAAGATG ATTAGTTCTG TGGCACCAAA ACCAAGAGAT CAGTTTTCCT GTGAGAAGTA

3761
AAGGAAGCAT TGTAGAAAAA TAGATGTGTT GAAGTCTACC GGTGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG

3841
CCTGGCTGAC CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT

3921
CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTACGC

4001
CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG TACATGACCT TATGGGACTT TCCTACTTGG

4081
CAGTACATCT ACGTATTAGT CATCGCTATT ACCATTGTGA TGCGGTTTTG GCAGTACATC AATGGGCGTG GATAGCGGTT

4161
TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC

4241
CAAAATGTCG TAACAACTCC GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT

4321
CGTTTAGTGA ACCGTCAGAT CTACCTCTTC CGCATCGCTG TCTGCGAGGG CCAGCTGTTG GGGTGAGTAC TCCCTCTCAA

4401
AAGCGGGCAT GACTTCTGCG CTAAGATTGT CAGTTTCCAA AAACGAGGAG GATTTGATAT TCACCTGGCC CGCGGTGATG

4481
CCTTTGAGGG TGGCCGCGTC CATCTGGTCA GAAAAGACAA TCTTTTTGTT GTCAAGCTTC CTTGATGATG TCATACTTAT

4561
CCTGTCCCTT TTTTTTCCAC AGCTCGCGGT TGAGGACAAA CTCTTCGCGG TCTTTCCAGT ACTCTTGGAT CGGAAACCCG

4641
TCGGCCTCCG AACGGTACTC CGCCACCGAG GGACCTGAGC GAGTCCGCAT CGACCGGATC GGAAAACCTC GGATCCGAAT

4721
TCATAGATAA CTGATCCAGT GCCCCTAACG TTACTGGCCG AAGCCGCTTG GAATAAGGCC GGTGTGCGTT TGTCTATATG

4801
TTATTTTCCA CCATATTGCC GTCTTTTGGC AATGTGAGGG CCCGGAAACC TGGCCCTGTC TTCTTGACGA GCATTCCTAG

4881
GGGTCTTTCC CCTCTCGCCA AAGGAATGCA AGGTCTGTTG AATGTCGTGA AGGAAGCAGT TCCTCTGGAA GCTTCTTGAA

4961
GACAAACAAC GTCTGTAGCG ACCCTTTGCA GGCAGCGGAA CCCCCCACCT GGCGACAGGT GCCTCTGCGG CCAAAAGCCA

5041
CGTGTATAAG ATACACCTGC AAAGGCGGCA CAACCCCAGT GCCACGTTGT GAGTTGGATA GTTGTGGAAA GAGTCAAATG

5121
GCTCTCCTCA AGCGTATTCA ACAAGGGGCT GAAGGATGCC CAGAAGGTAC CCCATTGTAT GGGATCTGAT CTGGGGCCTC

5201
GGTGCACATG CTTTACATGT GTTTAGTCGA GGTTAAAAAA CGTCTAGGCC CCCCGAACCA CGGGGACGTG GTTTTCCTTT

5281
GAAAAACACG ATGATAATAT GGCCACAACC ATGGCCACCT CAGCAAGTTC CCACTTGAAC AAAAACATCA AGCAAATGTA

5361
CTTGTGCCTG CCCCAGGGTG AGAAAGTCCA AGCCATGTAT ATCTGGGTTG ATGGTACTGG AGAAGGACTG CGCTGCAAAA

5441
CCCGCACCCT GGACTGTGAG CCCAAGTGTG TAGAAGAGTT ACCTGAGTGG AATTTTGATG GCTCTAGTAC CTTTCAGTCT

5521
GAGGGCTCCA ACAGTGACAT GTATCTCAGC CCTGTTGCCA TGTTTCGGGA CCCCTTCCGC AGAGATCCCA ACAAGCTGGT

5601
GTTCTGTGAA GTTTTCAAGT ACAACCGGAA GCCTGCAGAG ACCAATTTAA GGCACTCGTG TAAACGGATA ATGGACATGG

5681
TGAGCAACCA GCACCCCTGG TTTGGAATGG AACAGGAGTA TACTCTGATG GGAACAGATG GGCACCCTTT TGGTTGGCCT

5761
TCCAATGGCT TTCCTGGGCC CCAAGGTCCG TATTACTGTG GTGTGGGCGC AGACAAAGCC TATGGCAGGG ATATCGTGGA

5841
GGCTCACTAC CGCGCCTGCT TGTATGCTGG GGTCAAGATT ACAGGAACAA ATGCTGAGGT CATGCCTGCC CAGTGGGAGT

5921
TCCAAATAGG ACCCTGTGAA GGAATCCGCA TGGGAGATCA TCTCTGGGTG GCCCGTTTCA TCTTGCATCG AGTATGTGAA

6001
GACTTTGGGG TAATAGCAAC CTTTGACCCC AAGCCCATTC CTGGGAACTG GAATGGTGCA GGCTGCCATA CCAACTTTAG

6081
CACCAAGGCC ATGCGGGAGG AGAATGGTCT GAAGCACATC GAGGAGGCCA TCGAGAAACT AAGCAAGCGG CACCGGTACC

6161
ACATTCGAGC CTACGATCCC AAGGGGGGCC TGGACAATGC CCGTCGTCTG ACTGGGTTCC ACGAAACGTC CAACATCAAC

6241
GACTTTTCTG CTGGTGTCGC CAATCGCAGT GCCAGCATCC GCATTCCCCG GACTGTCGGC CAGGAGAAGA AAGGTTACTT

6321
TGAAGACCGC CGCCCCTCTG CCAATTGTGA CCCCTTTGCA GTGACAGAAG CCATCGTCCG CACATGCCTT CTCAATGAGA

6401
CTGGCGACGA GCCCTTCCAA TACAAAAACT AATCTAGATC CCCCTCGCTT TCTTGCTGTC CAATTTCTAT TAAAGGTTCC

6481
TTTGTTCCCT AAGTCCAACT ACTAAACTGG GGGATATTAT GAAGGGCCTT GAGCATCTGG ATTCTGCCTA ATAAAAAACA

6561
TTTATTTTCA TTGCAATGAT GTATTTAAAT TATTTCTGAA TATTTTACTA AAAAGGGAAT GTGGGAGGTC AGTGCATTTA

6641
AAACATAAAG AAATGAAGAG GGGGATCTTC GCGACCTGCA GGTCTCCCTA TAGTGAGTCG TATTAATTTC GATAAGCCAG

6721
CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC

6801
GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG

6881
ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC

6961
CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG

7041
ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT

7121
TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG

7201
CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT

7281
AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT

7361
TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC

7441
GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT

7521
TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC

7601
GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA

7681
ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT

7761
ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG

7841
CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC

7921
AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT

8001
TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT

8081
CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC

8161
GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC

8241
CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT

8321
GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG

8401
CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC

8481
TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA

8561
AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA

8641
TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TCTAAGAAAC

8721
CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC ACGAGGCCCT TTCGTCTCGC GCGTTTCGGT GATGACGGTG

8801
AAAACCTCTG ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA AGCCCGTCAG

8881
GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCTGGCTTA ACTATGCGGC ATCAGAGCAG ATTGTACTGA GAGTGCACCA

8961
TATCGACGCT CTCCCTTATG CGACTCCTGC ATTAGGAAGC AGCCCAGTAG TAGGTTGAGG CCGTTGAGCA CCGCCGCCGC

9041
AAGGAATGGT GCATGCAAGG AGATGGCGCC CAACAGTCCC CCGGCCACGG GGCCTGCCAC CATACCCACG CCGAAACAAG

9121
CGCTCATGAG CCCGAAGTGG CGAGCCCGAT CTTCCCCATC GGTGATGTCG GCGATATAGG CGCCAGCAAC CGCACCTGTG

9201
GCGCCGGTGA TGCCGGCCAC GATGCGTCCG GCGTAGAGGA TCTGGCTAGC GATGACCCTG CTGATTGGTT CGCTGACCAT

9281
TTCCGGGGTG CGGAACGGCG TTACCAGAAA CTCAGAAGGT TCGTCCAACC AAACCGACTC TGACGGCAGT TTACGAGAGA

9361
GATGATAGGG TCTGCTTCAG TAAGCCAGAT GCTACACAAT TAGGCTTGTA CATATTGTCG TTAGAACGCG GCTACAATTA

9441
ATACATAACC TTATGTATCA TACACATACG ATTTAGGTGA CACTATAGAA TACACCTGCA GGACGCGTAC TGAGAGCGCT

9521
ATTCTGAACT TTTCTTTTGT TC

pCLD22 (SEQ ID NO: 6):

1
CCTTCCCTTC TACCACACCC TAATTGTAAT CCATTTTAAT TTCCTGGTCA CAGTCCTGTC TCTCCTTCCA TTGTACCTTG

81
CCCTTTTCTA AAGAGCGACT GCAAAGTATG TTTGCGTAGG TGAGGATCTA AAACTTTATG AGGTACGAAC ATCACAGAAT

161
TACTTTGTAA TTTCAGTTTA TTGTAGGCTT GGCTTTTTGG GGAGGGTTTA CGTCTTAGAC CTCTTAGTGC TTCTTTGTTT

241
CATGGTGTTC TAACTTCGAA GCATCTCTGT AGCTTTAATG GATTCCTTTT CTGAAAGCTT TGCTCTCTTT CTTCCCCCTC

321
GGCTTTCTCT TAGGCAAGAG GGCTAACTGT AAAGTAAGGC TTACTGCCTT GTGTTTCCAA ATGTGTCCGA AGAGGAAGTG

401
TCTTCTGTGA ATCCTGTTAT GCATGAATAA CAGGAAATAG AAAGAAATTC ACTTTCATTA TTATAAAAGT AATATGTTCG

481
TTTAAAAAAT TCTAATGAAG AGCTGGAGAT GCAACCCAGG GGTAGAGCAC ACACTCAGCA TGCAGGAGGC CCTGGGTCCA

561
ATCTTGGAAT CTCCTCTCAG TTAACCTGAT CTCTAGCTGA TTAGTAGTGA GTGCAAGCCC ACTTTCCTCT TCTGCCTCAT

641
TGCTCAGTGA TAACAGCTGT TAAACTTTGT CTTATTCTAA AACTACCTCT GTGCAAATGC TAGCACAATA ATATATATCA

721
TATGCACATG ATTTTTTTTT TATCTTGAAA AGTAAGTCAG TATAGCTACA AAGTTCACTT GGCATTGTCA ACATTTCACA

801
GGCGTAATAT TCCTCCTCTA GTACTGTCCT CTTCATTCTT TGTGACCAAG TTTGGAGAGA GTGCACAAAT GCCAGGGAGG

881
TTTGTGGGAA GGTTTCTCAT GTTCTGGTAA GGCGAGTAAG AAAATAGTCT CATGCAGGTG AAATGAGTGC TATGCAGTAT

961
ATATTATACC AGAGAACAGC AAATGACCAA ATTCACACTG AACTAGTTCA GTAAAATTGG CTTTGTCAAA GCTTTCCTTG

1041
CTTAAAATGT AATTCCCTGT CATCCTAGTT CTGGTCTGGA TTCTTTTCCT GGAGTCTTGA CTTCCAGATT CCCTGTGGAC

1121
TTTTGTTTGA GTTTCAAGCT TTTGAAATAT AGAAACCTAT CTAACTTAAC AAACTTGGGA GAGAAAAGAC TCCAGAACAA

1201
CTGAAAACAG ACCAGGCTAA ATGAATAGAC TTTATTCCTC TCTTCTTACC TGCAGTTTTC AGATATGCAG AGTTGGAGCG

1281
GATCTTAGAG GTTGATTCAT TCATGCCTGA AGAAAACACA TTTTATAGAC CCTGTGCCCA AGTTCGTGGT GGACATCACC

1361
CTTTATTTAC TAATTGCACT ACATAACAGG CATTTTAGAA GACTGCTCCA GTCAGAGACC CCGCCTTAGA GGAATCTGTA

1441
AACCCTGAAC TCCTATCACT CATGAGCACT AGTTATGTTT GGAATGCCGT ATTAAAACAA AAGTTACATT TCTAAACTTA

1521
AAATTTTCTA GCACAGAGAC AGTGGGAGTA GCTAACTTTG ATAGACATTT TTCTACTAAA AGTCTTTCTA AGTACATAAT

1601
CTTCTGTAAG TTGGAAAACA GCAAAATAGA ACGTCTCCTA CGTAGTTAAT CTTTTTGCAT AATTTGCACA TGTAGGAGTT

1681
ATTAGTATAC GGGTAAGTTT TCACTTTTTC CCCCAACTGG AGTGTCTTGT GGCTGGGTTT GAAAAAGGGA ACGGGAGGCC

1761
GCTGGAGGGG ATTGGTAAAT GAGATAAAAC ACCACTCATT CAACTCAGTG ACTCAGCATT TAAATTTTCC ATAAAAGGAT

1841
TAAAGGAAAA TTAAACAAAT TCTTAAAGCC AAGACTCTGG AGAAACTTGT TGGTGTGCTT TAGTTTTCAC TGTTATGACT

1921
CATGAATTTA TGCATAAATT AGTACATTTA TAAAAACATA GCCTTTTTAG AGTTTTCTGT TTGGCTAAAG TGCCATTGTT

2001
AGCATTTGGA ATTACCTTTT TATGTCTTAT ATTTTTTCCA AATAAAAATA AATGTTTCTG CTGTCTTACT ACTGAAACTA

2081
CGTTGTGAGC ACTTTAAATT TCTCAAAGCA GTTTCGCCTG TTATACTTGG CGCTTAGTCA TCGTCGTACA CAACAGGACC

2161
TGATTAAGAA GGCTGTGCTG CCTCTAAGCC GGGCTAGATT GTAGCCACTA GCAACCAGGC TGCAATAATT TCCCTTTGAT

2241
GACATCATCC ACTGTGGAAG AACCCAGTTG CTTCAGCCAG TCGAACTATA CAGTTCCAAC CTCATCAAAT ATGGCATCTC

2321
CCTTGCCTGC TATAGCAGGG GGAGGAAAAA ATGCCACCAT CTTTTTAATC TAGCAAGCTT CTCTTTTCTT CATCTTTTTT

2401
TTTTTCTTTT AAAAAAATTC TGATCATGGA TGCTTCTTCC GATCCCTATT TGCCTTATGA CGGGGGAGGA GACAATATCC

2481
CCTTGAGGGA ATTACATAAA AGAGGTAAGA GCATCCCCTT GCTCTGAATC CTCTGTTGGT TGTTGTGCAT GCGGCTGGGC

2561
GGTTCTGGGG ACAGGCTGTC TGTTGTCCTC TTGCTGCAAT GTGCTGCTTA GTTGCCCTGC CTTGTTGCTG TGGGAGAATG

2641
CGACCTTCCC AGCAGGGCTG GCCCTCCCTG ATTGTTTGCT CTGTGCAGAT TAGCCCTGCT TCAGATCACA TAGGGCTGCA

2721
GACTCCATCT TCTGTGTGAA AATGCTTTCG GTTTGATTGC AGAAATAAGC TGCCTTTACA GCCAGCTAAA GTCCTGGTGG

2801
TTGGTTGGCA CCTGCAAAGT AGTATTTTTG TACCTCTGGA AACTTATATT TTCTTTACAC AGCAATATCA AGTGCCGGTA

2881
TGCCATTCTG TTTTGGCTGC TGCCAATTAC CATGTAGACT TTGCACCACA GAGTAATAGT AAAAGCTCCT AGCTGCATTT

2961
TATAACATTT AAAAATAGCA GGAAAGAAGA ATTATTTTTG ATTTAACATG TTTTTGTCAT TTAACGTCTT AACTGATTGA

3041
CATACTATAT TGTCTGTCTC GTGGGTATCT TGTACAACTT GATAGGATAA AGCAATTTAG TTTTTTTTTT TTTTTTTAAA

3121
TACATCCAGA ATGTAAGTCG TCAGTAGTTT TCGAACAGAT AAGTAATGGT GTTAATCTTT TGGCAGGCTT TGCCTTGGTC

3201
TCCTTAAAGC TAATTAGGTG TTACTTAATT AAACTGCTCT TTTGCTCATT TTCTTAAATT ATTTTTTTAA AAGATAGTTG

3281
GCATTTGCTG TTCTAGAAAT AAACTTCAAG AAACATTCTT TAGCCAGATG ACTTCATGTA TGAGCCATGT TAGTTTGAAT

3361
TATTTGCTTG GTGTTATAAA CTTTATGGTT TAATACCAAC TTTTATTATG TTTACAAGGT AAATAAGGAA AATTTCAAGT

3441
ACATTTTGTA TCCTGAGAAC AAATTTAAGT TCCATAGAAT TTAGGAATTA CAATGTATTC AACAGATACT TACTTGTCAT

3521
ACTGTGCCTG CAAAACAATA ATTAGACTCT GAACAGGTGC AACAATTTTC TGTAGAATTA GACAAGTCTT CTTTTGGCAG

3601
GTGTTACTAA GTAGGCCATT TCCCAAGGAA CAGGGAATTT GCCAGGCTTT TGTGGTGGAG AGAATAGAAT GAATAAATGC

3681
TGTGGGGAGT AAAGAGCTTG TCAGAAGATG ATTAGTTCTG TGGCACCAAA ACCAAGAGAT CAGTTTTCCT GTGAGAAGTA

3761
AAGGAAGCAT TGTAGAAAAA TAGATGTGTT GAAGTCTACC GGTGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG

3841
CCTGGCTGAC CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT

3921
CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTACGC

4001
CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG TACATGACCT TATGGGACTT TCCTACTTGG

4081
CAGTACATCT ACGTATTAGT CATCGCTATT ACCATTGTGA TGCGGTTTTG GCAGTACATC AATGGGCGTG GATAGCGGTT

4161
TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC

4241
CAAAATGTCG TAACAACTCC GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT

4321
CGTTTAGTGA ACCGTCAGAT CTACCTCTTC CGCATCGCTG TCTGCGAGGG CCAGCTGTTG GGGTGAGTAC TCCCTCTCAA

4401
AAGCGGGCAT GACTTCTGCG CTAAGATTGT CAGTTTCCAA AAACGAGGAG GATTTGATAT TCACCTGGCC CGCGGTGATG

4481
CCTTTGAGGG TGGCCGCGTC CATCTGGTCA GAAAAGACAA TCTTTTTGTT GTCAAGCTTC CTTGATGATG TCATACTTAT

4561
CCTGTCCCTT TTTTTTCCAC AGCTCGCGGT TGAGGACAAA CTCTTCGCGG TCTTTCCAGT ACTCTTGGAT CGGAAACCCG

4641
TCGGCCTCCG AACGGTACTC CGCCACCGAG GGACCTGAGC GAGTCCGCAT CGACCGGATC GGAAAACCTC GGATCCGAAT

4721
TCATAGATAA CTGATCCAGT GCCCCTAACG TTACTGGCCG AAGCCGCTTG GAATAAGGCC GGTGTGCGTT TGTCTATATG

4801
TTATTTTCCA CCATATTGCC GTCTTTTGGC AATGTGAGGG CCCGGAAACC TGGCCCTGTC TTCTTGACGA GCATTCCTAG

4881
GGGTCTTTCC CCTCTCGCCA AAGGAATGCA AGGTCTGTTG AATGTCGTGA AGGAAGCAGT TCCTCTGGAA GCTTCTTGAA

4961
GACAAACAAC GTCTGTAGCG ACCCTTTGCA GGCAGCGGAA CCCCCCACCT GGCGACAGGT GCCTCTGCGG CCAAAAGCCA

5041
CGTGTATAAG ATACACCTGC AAAGGCGGCA CAACCCCAGT GCCACGTTGT GAGTTGGATA GTTGTGGAAA GAGTCAAATG

5121
GCTCTCCTCA AGCGTATTCA ACAAGGGGCT GAAGGATGCC CAGAAGGTAC CCCATTGTAT GGGATCTGAT CTGGGGCCTC

5201
GGTGCACATG CTTTACATGT GTTTAGTCGA GGTTAAAAAA CGTCTAGGCC CCCCGAACCA CGGGGACGTG GTTTTCCTTT

5281
GAAAAACACG ATGGCCACCT CAGCAAGTTC CCACTTGAAC AAAAACATCA AGCAAATGTA CTTGTGCCTG CCCCAGGGTG

5361
AGAAAGTCCA AGCCATGTAT ATCTGGGTTG ATGGTACTGG AGAAGGACTG CGCTGCAAAA CCCGCACCCT GGACTGTGAG

5441
CCCAAGTGTG TAGAAGAGTT ACCTGAGTGG AATTTTGATG GCTCTAGTAC CTTTCAGTCT GAGGGCTCCA ACAGTGACAT

5521
GTATCTCAGC CCTGTTGCCA TGTTTCGGGA CCCCTTCCGC AGAGATCCCA ACAAGCTGGT GTTCTGTGAA GTTTTCAAGT

5601
ACAACCGGAA GCCTGCAGAG ACCAATTTAA GGCACTCGTG TAAACGGATA ATGGACATGG TGAGCAACCA GCACCCCTGG

5681
TTTGGAATGG AACAGGAGTA TACTCTGATG GGAACAGATG GGCACCCTTT TGGTTGGCCT TCCAATGGCT TTCCTGGGCC

5761
CCAAGGTCCG TATTACTGTG GTGTGGGCGC AGACAAAGCC TATGGCAGGG ATATCGTGGA GGCTCACTAC CGCGCCTGCT

5841
TGTATGCTGG GGTCAAGATT ACAGGAACAA ATGCTGAGGT CATGCCTGCC CAGTGGGAGT TCCAAATAGG ACCCTGTGAA

5921
GGAATCCGCA TGGGAGATCA TCTCTGGGTG GCCCGTTTCA TCTTGCATCG AGTATGTGAA GACTTTGGGG TAATAGCAAC

6001
CTTTGACCCC AAGCCCATTC CTGGGAACTG GAATGGTGCA GGCTGCCATA CCAACTTTAG CACCAAGGCC ATGCGGGAGG

6081
AGAATGGTCT GAAGCACATC GAGGAGGCCA TCGAGAAACT AAGCAAGCGG CACCGGTACC ACATTCGAGC CTACGATCCC

6161
AAGGGGGGCC TGGACAATGC CCGTCGTCTG ACTGGGTTCC ACGAAACGTC CAACATCAAC GACTTTTCTG CTGGTGTCGC

6241
CAATCGCAGT GCCAGCATCC GCATTCCCCG GACTGTCGGC CAGGAGAAGA AAGGTTACTT TGAAGACCGC CGCCCCTCTG

6321
CCAATTGTGA CCCCTTTGCA GTGACAGAAG CCATCGTCCG CACATGCCTT CTCAATGAGA CTGGCGACGA GCCCTTCCAA

6401
TACAAAAACT AATCTAGATC CCCCTCGCTT TCTTGCTGTC CAATTTCTAT TAAAGGTTCC TTTGTTCCCT AAGTCCAACT

6481
ACTAAACTGG GGGATATTAT GAAGGGCCTT GAGCATCTGG ATTCTGCCTA ATAAAAAACA TTTATTTTCA TTGCAATGAT

6561
GTATTTAAAT TATTTCTGAA TATTTTACTA AAAAGGGAAT GTGGGAGGTC AGTGCATTTA AAACATAAAG AAATGAAGAG

6641
GGGGATCTTC GCGACCTGCA GGTCTCCCTA TAGTGAGTCG TATTAATTTC GATAAGCCAG CTGCATTAAT GAATCGGCCA

6721
ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC

6801
TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG

6881
TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA

6961
CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG

7041
GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG

7121
GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC

7201
CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC

7281
TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC

7361
TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC

7441
TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT

7521
CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG

7601
AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA

7681
AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG

7761
CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC

7841
CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT

7921
ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG

8001
TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG

8081
CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC

8161
CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA

8241
CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT

8321
AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT

8401
ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT

8481
CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC

8561
TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA

8641
TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TCTAAGAAAC CATTATTATC ATGACATTAA

8721
CCTATAAAAA TAGGCGTATC ACGAGGCCCT TTCGTCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG ACACATGCAG

8801
CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG

8881
CGGGTGTCGG GGCTGGCTTA ACTATGCGGC ATCAGAGCAG ATTGTACTGA GAGTGCACCA TATCGACGCT CTCCCTTATG

8961
CGACTCCTGC ATTAGGAAGC AGCCCAGTAG TAGGTTGAGG CCGTTGAGCA CCGCCGCCGC AAGGAATGGT GCATGCAAGG

9041
AGATGGCGCC CAACAGTCCC CCGGCCACGG GGCCTGCCAC CATACCCACG CCGAAACAAG CGCTCATGAG CCCGAAGTGG

9121
CGAGCCCGAT CTTCCCCATC GGTGATGTCG GCGATATAGG CGCCAGCAAC CGCACCTGTG GCGCCGGTGA TGCCGGCCAC

9201
GATGCGTCCG GCGTAGAGGA TCTGGCTAGC GATGACCCTG CTGATTGGTT CGCTGACCAT TTCCGGGGTG CGGAACGGCG

9281
TTACCAGAAA CTCAGAAGGT TCGTCCAACC AAACCGACTC TGACGGCAGT TTACGAGAGA GATGATAGGG TCTGCTTCAG

9361
TAAGCCAGAT GCTACACAAT TAGGCTTGTA CATATTGTCG TTAGAACGCG GCTACAATTA ATACATAACC TTATGTATCA

9441
TACACATACG ATTTAGGTGA CACTATAGAA TACACCTGCA GGACGCGTAC TGAGAGCGCT ATTCTGAACT TTTCTTTTGT

9521
TC

pCLD12 (SEQ ID NO: 7):

1
CCCTAGAAAG ATAATCATAT TGTGACGTAC GTTAAAGATA ATCATGCGTA AAATTGACGC ATGTGTTTTA TCGGTCTGTA

81
TATCGAGGTT TATTTATTAA TTTGAATAGA TATTAAGTTT TATTATATTT ACACTTACAT ACTAATAATA AATTCAACAA

161
ACAATTTATT TATGTTTATT TATTTATTAA AAAAAAACAA AAACTCAAAA TTTCTTCTAT AAAGTAACAA AACTTTTATC

241
GAATTTGCAG CCCGGGACTA GCTAGAGGGA CAGCCCCCCC CCAAAGCCCC CAGGGATGTA ATTACGTCCC TCCCCCGCTA

321
GGGGGCAGCA GCGAGCCGCC CGGGGCTCCG CTCCGGTCCG GCGCTCCCCC CGCATCCCCG AGCCGGCAGC GTGCGGGGAC

401
AGCCCGGGCA CGGGGAAGGT GGCACGGGAT CGCTTTCCTC TGAACGCTTC TCGCTGCTCT TTGAGCCTGC AGACACCTGG

481
GGGGATACGG GGAAAACTTA AGATCCGACC GGACGCGTAC TGAGAGCGCT ATTCTGAACT TTTCTTTTGT TCCCTTCCCT

561
TCTACCACAC CCTAATTGTA ATCCATTTTA ATTTCCTGGT CACAGTCCTG TCTCTCCTTC CATTGTACCT TGCCCTTTTC

641
TAAAGAGCGA CTGCAAAGTA TGTTTGCGTA GGTGAGGATC TAAAACTTTA TGAGGTACGA ACATCACAGA ATTACTTTGT

721
AATTTCAGTT TATTGTAGGC TTGGCTTTTT GGGGAGGGTT TACGTCTTAG ACCTCTTAGT GCTTCTTTGT TTCATGGTGT

801
TCTAACTTCG AAGCATCTCT GTAGCTTTAA TGGATTCCTT TTCTGAAAGC TTTGCTCTCT TTCTTCCCCC TCGGCTTTCT

881
CTTAGGCAAG AGGGCTAACT GTAAAGTAAG GCTTACTGCC TTGTGTTTCC AAATGTGTCC GAAGAGGAAG TGTCTTCTGT

961
GAATCCTGTT ATGCATGAAT AACAGGAAAT AGAAAGAAAT TCACTTTCAT TATTATAAAA GTAATATGTT CGTTTAAAAA

1041
ATTCTAATGA AGAGCTGGAG ATGCAACCCA GGGGTAGAGC ACACACTCAG CATGCAGGAG GCCCTGGGTC CAATCTTGGA

1121
ATCTCCTCTC AGTTAACCTG ATCTCTAGCT GATTAGTAGT GAGTGCAAGC CCACTTTCCT CTTCTGCCTC ATTGCTCAGT

1201
GATAACAGCT GTTAAACTTT GTCTTATTCT AAAACTACCT CTGTGCAAAT GCTAGCACAA TAATATATAT CATATGCACA

1281
TGATTTTTTT TTTATCTTGA AAAGTAAGTC AGTATAGCTA CAAAGTTCAC TTGGCATTGT CAACATTTCA CAGGCGTAAT

1361
ATTCCTCCTC TAGTACTGTC CTCTTCATTC TTTGTGACCA AGTTTGGAGA GAGTGCACAA ATGCCAGGGA GGTTTGTGGG

1441
AAGGTTTCTC ATGTTCTGGT AAGGCGAGTA AGAAAATAGT CTCATGCAGG TGAAATGAGT GCTATGCAGT ATATATTATA

1521
CCAGAGAACA GCAAATGACC AAATTCACAC TGAACTAGTT CAGTAAAATT GGCTTTGTCA AAGCTTTCCT TGCTTAAAAT

1601
GTAATTCCCT GTCATCCTAG TTCTGGTCTG GATTCTTTTC CTGGAGTCTT GACTTCCAGA TTCCCTGTGG ACTTTTGTTT

1681
GAGTTTCAAG CTTTTGAAAT ATAGAAACCT ATCTAACTTA ACAAACTTGG GAGAGAAAAG ACTCCAGAAC AACTGAAAAC

1761
AGACCAGGCT AAATGAATAG ACTTTATTCC TCTCTTCTTA CCTGCAGTTT TCAGATATGC AGAGTTGGAG CGGATCTTAG

1841
AGGTTGATTC ATTCATGCCT GAAGAAAACA CATTTTATAG ACCCTGTGCC CAAGTTCGTG GTGGACATCA CCCTTTATTT

1921
ACTAATTGCA CTACATAACA GGCATTTTAG AAGACTGCTC CAGTCAGAGA CCCCGCCTTA GAGGAATCTG TAAACCCTGA

2001
ACTCCTATCA CTCATGAGCA CTAGTTATGT TTGGAATGCC GTATTAAAAC AAAAGTTACA TTTCTAAACT TAAAATTTTC

2081
TAGCACAGAG ACAGTGGGAG TAGCTAACTT TGATAGACAT TTTTCTACTA AAAGTCTTTC TAAGTACATA ATCTTCTGTA

2161
AGTTGGAAAA CAGCAAAATA GAACGTCTCC TACGTAGTTA ATCTTTTTGC ATAATTTGCA CATGTAGGAG TTATTAGTAT

2241
ACGGGTAAGT TTTCACTTTT TCCCCCAACT GGAGTGTCTT GTGGCTGGGT TTGAAAAAGG GAACGGGAGG CCGCTGGAGG

2321
GGATTGGTAA ATGAGATAAA ACACCACTCA TTCAACTCAG TGACTCAGCA TTTAAATTTT CCATAAAAGG ATTAAAGGAA

2401
AATTAAACAA ATTCTTAAAG CCAAGACTCT GGAGAAACTT GTTGGTGTGC TTTAGTTTTC ACTGTTATGA CTCATGAATT

2481
TATGCATAAA TTAGTACATT TATAAAAACA TAGCCTTTTT AGAGTTTTCT GTTTGGCTAA AGTGCCATTG TTAGCATTTG

2561
GAATTACCTT TTTATGTCTT ATATTTTTTC CAAATAAAAA TAAATGTTTC TGCTGTCTTA CTACTGAAAC TACGTTGTGA

2641
GCACTTTAAA TTTCTCAAAG CAGTTTCGCC TGTTATACTT GGCGCTTAGT CATCGTCGTA CACAACAGGA CCTGATTAAG

2721
AAGGCTGTGC TGCCTCTAAG CCGGGCTAGA TTGTAGCCAC TAGCAACCAG GCTGCAATAA TTTCCCTTTG ATGACATCAT

2801
CCACTGTGGA AGAACCCAGT TGCTTCAGCC AGTCGAACTA TACAGTTCCA ACCTCATCAA ATATGGCATC TCCCTTGCCT

2881
GCTATAGCAG GGGGAGGAAA AAATGCCACC ATCTTTTTAA TCTAGCAAGC TTCTCTTTTC TTCATCTTTT TTTTTTTCTT

2961
TTAAAAAAAT TCTGATCATG GATGCTTCTT CCGATCCCTA TTTGCCTTAT GACGGGGGAG GAGACAATAT CCCCTTGAGG

3041
GAATTACATA AAAGAGGTAA GAGCATCCCC TTGCTCTGAA TCCTCTGTTG GTTGTTGTGC ATGCGGCTGG GCGGTTCTGG

3121
GGACAGGCTG TCTGTTGTCC TCTTGCTGCA ATGTGCTGCT TAGTTGCCCT GCCTTGTTGC TGTGGGAGAA TGCGACCTTC

3201
CCAGCAGGGC TGGCCCTCCC TGATTGTTTG CTCTGTGCAG ATTAGCCCTG CTTCAGATCA CATAGGGCTG CAGACTCCAT

3281
CTTCTGTGTG AAAATGCTTT CGGTTTGATT GCAGAAATAA GCTGCCTTTA CAGCCAGCTA AAGTCCTGGT GGTTGGTTGG

3361
CACCTGCAAA GTAGTATTTT TGTACCTCTG GAAACTTATA TTTTCTTTAC ACAGCAATAT CAAGTGCCGG TATGCCATTC

3441
TGTTTTGGCT GCTGCCAATT ACCATGTAGA CTTTGCACCA CAGAGTAATA GTAAAAGCTC CTAGCTGCAT TTTATAACAT

3521
TTAAAAATAG CAGGAAAGAA GAATTATTTT TGATTTAACA TGTTTTTGTC ATTTAACGTC TTAACTGATT GACATACTAT

3601
ATTGTCTGTC TCGTGGGTAT CTTGTACAAC TTGATAGGAT AAAGCAATTT AGTTTTTTTT TTTTTTTTTA AATACATCCA

3681
GAATGTAAGT CGTCAGTAGT TTTCGAACAG ATAAGTAATG GTGTTAATCT TTTGGCAGGC TTTGCCTTGG TCTCCTTAAA

3761
GCTAATTAGG TGTTACTTAA TTAAACTGCT CTTTTGCTCA TTTTCTTAAA TTATTTTTTT AAAAGATAGT TGGCATTTGC

3841
TGTTCTAGAA ATAAACTTCA AGAAACATTC TTTAGCCAGA TGACTTCATG TATGAGCCAT GTTAGTTTGA ATTATTTGCT

3921
TGGTGTTATA AACTTTATGG TTTAATACCA ACTTTTATTA TGTTTACAAG GTAAATAAGG AAAATTTCAA GTACATTTTG

4001
TATCCTGAGA ACAAATTTAA GTTCCATAGA ATTTAGGAAT TACAATGTAT TCAACAGATA CTTACTTGTC ATACTGTGCC

4081
TGCAAAACAA TAATTAGACT CTGAACAGGT GCAACAATTT TCTGTAGAAT TAGACAAGTC TTCTTTTGGC AGGTGTTACT

4161
AAGTAGGCCA TTTCCCAAGG AACAGGGAAT TTGCCAGGCT TTTGTGGTGG AGAGAATAGA ATGAATAAAT GCTGTGGGGA

4241
GTAAAGAGCT TGTCAGAAGA TGATTAGTTC TGTGGCACCA AAACCAAGAG ATCAGTTTTC CTGTGAGAAG TAAAGGAAGC

4321
ATTGTAGAAA AATAGATGTG TTGAAGTCTA CCGGTGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG

4401
ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC

4481
GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT

4561
GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT

4641
CTACGTATTA GTCATCGCTA TTACCATTGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC

4721
GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT

4801
CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCGTTTAGT

4881
GAACCGTCAG ATCTACCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGGTGAGT ACTCCCTCTC AAAAGCGGGC

4961
ATGACTTCTG CGCTAAGATT GTCAGTTTCC AAAAACGAGG AGGATTTGAT ATTCACCTGG CCCGCGGTGA TGCCTTTGAG

5041
GGTGGCCGCG TCCATCTGGT CAGAAAAGAC AATCTTTTTG TTGTCAAGCT TCCTTGATGA TGTCATACTT ATCCTGTCCC

5121
TTTTTTTTCC ACAGCTCGCG GTTGAGGACA AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC

5201
CGAACGGTAC TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCGGATCCGA ATTCATAGAT

5281
AACTGATCCA GTGCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG CCGGTGTGCG TTTGTCTATA TGTTATTTTC

5361
CACCATATTG CCGTCTTTTG GCAATGTGAG GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT

5441
CCCCTCTCGC CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG AAGACAAACA

5521
ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG GTGCCTCTGC GGCCAAAAGC CACGTGTATA

5601
AGATACACCT GCAAAGGCGG CACAACCCCA GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT

5681
CAAGCGTATT CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC TCGGTGCACA

5761
TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AACGTCTAGG CCCCCCGAAC CACGGGGACG TGGTTTTCCT TTGAAAAACA

5841
CGATGATAAT ATGGCCACAA CCATGGCCAC CTCAGCAAGT TCCCACTTGA ACAAAAACAT CAAGCAAATG TACTTGTGCC

5921
TGCCCCAGGG TGAGAAAGTC CAAGCCATGT ATATCTGGGT TGATGGTACT GGAGAAGGAC TGCGCTGCAA AACCCGCACC

6001
CTGGACTGTG AGCCCAAGTG TGTAGAAGAG TTACCTGAGT GGAATTTTGA TGGCTCTAGT ACCTTTCAGT CTGAGGGCTC

6081
CAACAGTGAC ATGTATCTCA GCCCTGTTGC CATGTTTCGG GACCCCTTCC GCAGAGATCC CAACAAGCTG GTGTTCTGTG

6161
AAGTTTTCAA GTACAACCGG AAGCCTGCAG AGACCAATTT AAGGCACTCG TGTAAACGGA TAATGGACAT GGTGAGCAAC

6241
CAGCACCCCT GGTTTGGAAT GGAACAGGAG TATACTCTGA TGGGAACAGA TGGGCACCCT TTTGGTTGGC CTTCCAATGG

6321
CTTTCCTGGG CCCCAAGGTC CGTATTACTG TGGTGTGGGC GCAGACAAAG CCTATGGCAG GGATATCGTG GAGGCTCACT

6401
ACCGCGCCTG CTTGTATGCT GGGGTCAAGA TTACAGGAAC AAATGCTGAG GTCATGCCTG CCCAGTGGGA GTTCCAAATA

6481
GGACCCTGTG AAGGAATCCG CATGGGAGAT CATCTCTGGG TGGCCCGTTT CATCTTGCAT CGAGTATGTG AAGACTTTGG

6561
GGTAATAGCA ACCTTTGACC CCAAGCCCAT TCCTGGGAAC TGGAATGGTG CAGGCTGCCA TACCAACTTT AGCACCAAGG

6641
CCATGCGGGA GGAGAATGGT CTGAAGCACA TCGAGGAGGC CATCGAGAAA CTAAGCAAGC GGCACCGGTA CCACATTCGA

6721
GCCTACGATC CCAAGGGGGG CCTGGACAAT GCCCGTCGTC TGACTGGGTT CCACGAAACG TCCAACATCA ACGACTTTTC

6801
TGCTGGTGTC GCCAATCGCA GTGCCAGCAT CCGCATTCCC CGGACTGTCG GCCAGGAGAA GAAAGGTTAC TTTGAAGACC

6881
GCCGCCCCTC TGCCAATTGT GACCCCTTTG CAGTGACAGA AGCCATCGTC CGCACATGCC TTCTCAATGA GACTGGCGAC

6961
GAGCCCTTCC AATACAAAAA CTAATCTAGA TCCCCCTCGC TTTCTTGCTG TCCAATTTCT ATTAAAGGTT CCTTTGTTCC

7041
CTAAGTCCAA CTACTAAACT GGGGGATATT ATGAAGGGCC TTGAGCATCT GGATTCTGCC TAATAAAAAA CATTTATTTT

7121
CATTGCAATG ATGTATTTAA ATTATTTCTG AATATTTTAC TAAAAAGGGA ATGTGGGAGG TCAGTGCATT TAAAACATAA

7201
AGAAATGAAG AGGGGGATCT TCGCGATACT GCATCGATGA GGGACAGCCC CCCCCCAAAG CCCCCAGGGA TGTAATTACG

7281
TCCCTCCCCC GCTAGGGGGC AGCAGCGAGC CGCCCGGGGC TCCGCTCCGG TCCGGCGCTC CCCCCGCATC CCCGAGCCGG

7361
CAGCGTGCGG GGACAGCCCG GGCACGGGGA AGGTGGCACG GGATCGCTTT CCTCTGAACG CTTCTCGCTG CTCTTTGAGC

7441
CTGCAGACAC CTGGGGGGAT ACGGGGAAAA TAGACACCGC GGTGGAGCTC CAGCTTTTGT TCCCTTTAGT GAGGGTTAAT

7521
TAGTTCTTAA TACGACTCAC TATAGGGCGA ATTGGCTACC GGGCCGCCCA TCGAGGGTAT CATAAGCTTA TATCTATAAC

7601
AAGAAAATAT ATATATAATA AGTTATCACG TAAGTAGAAC ATGAAATAAC AATATAATTA TCGTATGAGT TAAATCTTAA

7681
AAGTCACGTA AAAGATAATC ATGCGTCATT TTGACTCACG CGGTCGTTAT AGTTCAAAAT CAGTGACACT TACCGCATTG

7761
ACAAGCACGC CTCACGGGAG CTCCAAGCGG CGACTGAGAT GTCCTAAATG CACAGCGACG GATTCGCGCT ATTTAGAAAG

7841
AGAGAGCAAT ATTTCAAGAA TGCATGCGTC AATTTTACGC AGACTATCTT TCTAGGGTTA AATCGATAGA TGCGATCCTG

7921
CAGGTCTCCC TATAGTGAGT CGTATTAATT TCGATAAGCC AGCTGCATTA ATGAATCGGC CAACGCGCGG GGAGAGGCGG

8001
TTTGCGTATT GGGCGCTCTT CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG

8081
CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA

8161
AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG

8241
ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT

8321
CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA

8401
CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG

8481
CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA

8561
ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG

8641
ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC

8721
CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA

8801
TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC

8881
TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA

8961
CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT

9041
AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA

9121
GATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC

9201
TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA

9281
TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC

9361
ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT

9441
GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA

9521
AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG ATAATACCGC GCCACATAGC

9601
AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG

9681
TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG

9761
GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT

9841
TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC

9921
GCGCACATTT CCCCGAAAAG TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT AACCTATAAA AATAGGCGTA

10001
TCACGAGGCC CTTTCGTCTC GCGCGTTTCG GTGATGACGG TGAAAACCTC TGACACATGC AGCTCCCGGA GACGGTCACA

10081
GCTTGTCTGT AAGCGGATGC CGGGAGCAGA CAAGCCCGTC AGGGCGCGTC AGCGGGTGTT GGCGGGTGTC GGGGCTGGCT

10161
TAACTATGCG GCATCAGAGC AGATTGTACT GAGAGTGCAC CATATCGACG CTCTCCCTTA TGCGACTCCT GCATTAGGAA

10241
GCAGCCCAGT AGTAGGTTGA GGCCGTTGAG CACCGCCGCC GCAAGGAATG GTGCATGCAA GGAGATGGCG CCCAACAGTC

10321
CCCCGGCCAC GGGGCCTGCC ACCATACCCA CGCCGAAACA AGCGCTCATG AGCCCGAAGT GGCGAGCCCG ATCTTCCCCA

10401
TCGGTGATGT CGGCGATATA GGCGCCAGCA ACCGCACCTG TGGCGCCGGT GATGCCGGCC ACGATGCGTC CGGCGTAGAG

10481
GATCTGGCTA GCGATGACCC TGCTGATTGG TTCGCTGACC ATTTCCGGGG TGCGGAACGG CGTTACCAGA AACTCAGAAG

10561
GTTCGTCCAA CCAAACCGAC TCTGACGGCA GTTTACGAGA GAGATGATAG GGTCTGCTTC AGTAAGCCAG ATGCTACACA

10641
ATTAGGCTTG TACATATTGT CGTTAGAACG CGGCTACAAT TAATACATAA CCTTATGTAT CATACACATA CGATTTAGGT

10721
GACACTATAG AATACACCTG CAGGACGTCC CAATGATCTT AAGTTAA

pCLD14 (SEQ ID NO: 8):

1
CCCTAGAAAG ATAATCATAT TGTGACGTAC GTTAAAGATA ATCATGCGTA AAATTGACGC ATGTGTTTTA TCGGTCTGTA

81
TATCGAGGTT TATTTATTAA TTTGAATAGA TATTAAGTTT TATTATATTT ACACTTACAT ACTAATAATA AATTCAACAA

161
ACAATTTATT TATGTTTATT TATTTATTAA AAAAAAACAA AAACTCAAAA TTTCTTCTAT AAAGTAACAA AACTTTTATC

241
GAATTTGCAG CCCGGGACTA GCTAGAGGGA CAGCCCCCCC CCAAAGCCCC CAGGGATGTA ATTACGTCCC TCCCCCGCTA

321
GGGGGCAGCA GCGAGCCGCC CGGGGCTCCG CTCCGGTCCG GCGCTCCCCC CGCATCCCCG AGCCGGCAGC GTGCGGGGAC

401
AGCCCGGGCA CGGGGAAGGT GGCACGGGAT CGCTTTCCTC TGAACGCTTC TCGCTGCTCT TTGAGCCTGC AGACACCTGG

481
GGGGATACGG GGAAAACTTA AGATCCGACC GGACGCGTAC TGAGAGCGCT ATTCTGAACT TTTCTTTTGT TCCCTTCCCT

561
TCTACCACAC CCTAATTGTA ATCCATTTTA ATTTCCTGGT CACAGTCCTG TCTCTCCTTC CATTGTACCT TGCCCTTTTC

641
TAAAGAGCGA CTGCAAAGTA TGTTTGCGTA GGTGAGGATC TAAAACTTTA TGAGGTACGA ACATCACAGA ATTACTTTGT

721
AATTTCAGTT TATTGTAGGC TTGGCTTTTT GGGGAGGGTT TACGTCTTAG ACCTCTTAGT GCTTCTTTGT TTCATGGTGT

801
TCTAACTTCG AAGCATCTCT GTAGCTTTAA TGGATTCCTT TTCTGAAAGC TTTGCTCTCT TTCTTCCCCC TCGGCTTTCT

881
CTTAGGCAAG AGGGCTAACT GTAAAGTAAG GCTTACTGCC TTGTGTTTCC AAATGTGTCC GAAGAGGAAG TGTCTTCTGT

961
GAATCCTGTT ATGCATGAAT AACAGGAAAT AGAAAGAAAT TCACTTTCAT TATTATAAAA GTAATATGTT CGTTTAAAAA

1041
ATTCTAATGA AGAGCTGGAG ATGCAACCCA GGGGTAGAGC ACACACTCAG CATGCAGGAG GCCCTGGGTC CAATCTTGGA

1121
ATCTCCTCTC AGTTAACCTG ATCTCTAGCT GATTAGTAGT GAGTGCAAGC CCACTTTCCT CTTCTGCCTC ATTGCTCAGT

1201
GATAACAGCT GTTAAACTTT GTCTTATTCT AAAACTACCT CTGTGCAAAT GCTAGCACAA TAATATATAT CATATGCACA

1281
TGATTTTTTT TTTATCTTGA AAAGTAAGTC AGTATAGCTA CAAAGTTCAC TTGGCATTGT CAACATTTCA CAGGCGTAAT

1361
ATTCCTCCTC TAGTACTGTC CTCTTCATTC TTTGTGACCA AGTTTGGAGA GAGTGCACAA ATGCCAGGGA GGTTTGTGGG

1441
AAGGTTTCTC ATGTTCTGGT AAGGCGAGTA AGAAAATAGT CTCATGCAGG TGAAATGAGT GCTATGCAGT ATATATTATA

1521
CCAGAGAACA GCAAATGACC AAATTCACAC TGAACTAGTT CAGTAAAATT GGCTTTGTCA AAGCTTTCCT TGCTTAAAAT

1601
GTAATTCCCT GTCATCCTAG TTCTGGTCTG GATTCTTTTC CTGGAGTCTT GACTTCCAGA TTCCCTGTGG ACTTTTGTTT

1681
GAGTTTCAAG CTTTTGAAAT ATAGAAACCT ATCTAACTTA ACAAACTTGG GAGAGAAAAG ACTCCAGAAC AACTGAAAAC

1761
AGACCAGGCT AAATGAATAG ACTTTATTCC TCTCTTCTTA CCTGCAGTTT TCAGATATGC AGAGTTGGAG CGGATCTTAG

1841
AGGTTGATTC ATTCATGCCT GAAGAAAACA CATTTTATAG ACCCTGTGCC CAAGTTCGTG GTGGACATCA CCCTTTATTT

1921
ACTAATTGCA CTACATAACA GGCATTTTAG AAGACTGCTC CAGTCAGAGA CCCCGCCTTA GAGGAATCTG TAAACCCTGA

2001
ACTCCTATCA CTCATGAGCA CTAGTTATGT TTGGAATGCC GTATTAAAAC AAAAGTTACA TTTCTAAACT TAAAATTTTC

2081
TAGCACAGAG ACAGTGGGAG TAGCTAACTT TGATAGACAT TTTTCTACTA AAAGTCTTTC TAAGTACATA ATCTTCTGTA

2161
AGTTGGAAAA CAGCAAAATA GAACGTCTCC TACGTAGTTA ATCTTTTTGC ATAATTTGCA CATGTAGGAG TTATTAGTAT

2241
ACGGGTAAGT TTTCACTTTT TCCCCCAACT GGAGTGTCTT GTGGCTGGGT TTGAAAAAGG GAACGGGAGG CCGCTGGAGG

2321
GGATTGGTAA ATGAGATAAA ACACCACTCA TTCAACTCAG TGACTCAGCA TTTAAATTTT CCATAAAAGG ATTAAAGGAA

2401
AATTAAACAA ATTCTTAAAG CCAAGACTCT GGAGAAACTT GTTGGTGTGC TTTAGTTTTC ACTGTTATGA CTCATGAATT

2481
TATGCATAAA TTAGTACATT TATAAAAACA TAGCCTTTTT AGAGTTTTCT GTTTGGCTAA AGTGCCATTG TTAGCATTTG

2561
GAATTACCTT TTTATGTCTT ATATTTTTTC CAAATAAAAA TAAATGTTTC TGCTGTCTTA CTACTGAAAC TACGTTGTGA

2641
GCACTTTAAA TTTCTCAAAG CAGTTTCGCC TGTTATACTT GGCGCTTAGT CATCGTCGTA CACAACAGGA CCTGATTAAG

2721
AAGGCTGTGC TGCCTCTAAG CCGGGCTAGA TTGTAGCCAC TAGCAACCAG GCTGCAATAA TTTCCCTTTG ATGACATCAT

2801
CCACTGTGGA AGAACCCAGT TGCTTCAGCC AGTCGAACTA TACAGTTCCA ACCTCATCAA ATATGGCATC TCCCTTGCCT

2881
GCTATAGCAG GGGGAGGAAA AAATGCCACC ATCTTTTTAA TCTAGCAAGC TTCTCTTTTC TTCATCTTTT TTTTTTTCTT

2961
TTAAAAAAAT TCTGATCATG GATGCTTCTT CCGATCCCTA TTTGCCTTAT GACGGGGGAG GAGACAATAT CCCCTTGAGG

3041
GAATTACATA AAAGAGGTAA GAGCATCCCC TTGCTCTGAA TCCTCTGTTG GTTGTTGTGC ATGCGGCTGG GCGGTTCTGG

3121
GGACAGGCTG TCTGTTGTCC TCTTGCTGCA ATGTGCTGCT TAGTTGCCCT GCCTTGTTGC TGTGGGAGAA TGCGACCTTC

3201
CCAGCAGGGC TGGCCCTCCC TGATTGTTTG CTCTGTGCAG ATTAGCCCTG CTTCAGATCA CATAGGGCTG CAGACTCCAT

3281
CTTCTGTGTG AAAATGCTTT CGGTTTGATT GCAGAAATAA GCTGCCTTTA CAGCCAGCTA AAGTCCTGGT GGTTGGTTGG

3361
CACCTGCAAA GTAGTATTTT TGTACCTCTG GAAACTTATA TTTTCTTTAC ACAGCAATAT CAAGTGCCGG TATGCCATTC

3441
TGTTTTGGCT GCTGCCAATT ACCATGTAGA CTTTGCACCA CAGAGTAATA GTAAAAGCTC CTAGCTGCAT TTTATAACAT

3521
TTAAAAATAG CAGGAAAGAA GAATTATTTT TGATTTAACA TGTTTTTGTC ATTTAACGTC TTAACTGATT GACATACTAT

3601
ATTGTCTGTC TCGTGGGTAT CTTGTACAAC TTGATAGGAT AAAGCAATTT AGTTTTTTTT TTTTTTTTTA AATACATCCA

3681
GAATGTAAGT CGTCAGTAGT TTTCGAACAG ATAAGTAATG GTGTTAATCT TTTGGCAGGC TTTGCCTTGG TCTCCTTAAA

3761
GCTAATTAGG TGTTACTTAA TTAAACTGCT CTTTTGCTCA TTTTCTTAAA TTATTTTTTT AAAAGATAGT TGGCATTTGC

3841
TGTTCTAGAA ATAAACTTCA AGAAACATTC TTTAGCCAGA TGACTTCATG TATGAGCCAT GTTAGTTTGA ATTATTTGCT

3921
TGGTGTTATA AACTTTATGG TTTAATACCA ACTTTTATTA TGTTTACAAG GTAAATAAGG AAAATTTCAA GTACATTTTG

4001
TATCCTGAGA ACAAATTTAA GTTCCATAGA ATTTAGGAAT TACAATGTAT TCAACAGATA CTTACTTGTC ATACTGTGCC

4081
TGCAAAACAA TAATTAGACT CTGAACAGGT GCAACAATTT TCTGTAGAAT TAGACAAGTC TTCTTTTGGC AGGTGTTACT

4161
AAGTAGGCCA TTTCCCAAGG AACAGGGAAT TTGCCAGGCT TTTGTGGTGG AGAGAATAGA ATGAATAAAT GCTGTGGGGA

4241
GTAAAGAGCT TGTCAGAAGA TGATTAGTTC TGTGGCACCA AAACCAAGAG ATCAGTTTTC CTGTGAGAAG TAAAGGAAGC

4321
ATTGTAGAAA AATAGATGTG TTGAAGTCTA CCGGTGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG

4401
ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC

4481
GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT

4561
GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT

4641
CTACGTATTA GTCATCGCTA TTACCATTGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC

4721
GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT

4801
CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCGTTTAGT

4881
GAACCGTCAG ATCTACCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGGTGAGT ACTCCCTCTC AAAAGCGGGC

4961
ATGACTTCTG CGCTAAGATT GTCAGTTTCC AAAAACGAGG AGGATTTGAT ATTCACCTGG CCCGCGGTGA TGCCTTTGAG

5041
GGTGGCCGCG TCCATCTGGT CAGAAAAGAC AATCTTTTTG TTGTCAAGCT TCCTTGATGA TGTCATACTT ATCCTGTCCC

5121
TTTTTTTTCC ACAGCTCGCG GTTGAGGACA AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC

5201
CGAACGGTAC TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCGGATCCGA ATTCATAGAT

5281
AACTGATCCA GTGCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG CCGGTGTGCG TTTGTCTATA TGTTATTTTC

5361
CACCATATTG CCGTCTTTTG GCAATGTGAG GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT

5441
CCCCTCTCGC CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG AAGACAAACA

5521
ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG GTGCCTCTGC GGCCAAAAGC CACGTGTATA

5601
AGATACACCT GCAAAGGCGG CACAACCCCA GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT

5681
CAAGCGTATT CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC TCGGTGCACA

5761
TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AACGTCTAGG CCCCCCGAAC CACGGGGACG TGGTTTTCCT TTGAAAAACA

5841
CGATGGCCAC CTCAGCAAGT TCCCACTTGA ACAAAAACAT CAAGCAAATG TACTTGTGCC TGCCCCAGGG TGAGAAAGTC

5921
CAAGCCATGT ATATCTGGGT TGATGGTACT GGAGAAGGAC TGCGCTGCAA AACCCGCACC CTGGACTGTG AGCCCAAGTG

6001
TGTAGAAGAG TTACCTGAGT GGAATTTTGA TGGCTCTAGT ACCTTTCAGT CTGAGGGCTC CAACAGTGAC ATGTATCTCA

6081
GCCCTGTTGC CATGTTTCGG GACCCCTTCC GCAGAGATCC CAACAAGCTG GTGTTCTGTG AAGTTTTCAA GTACAACCGG

6161
AAGCCTGCAG AGACCAATTT AAGGCACTCG TGTAAACGGA TAATGGACAT GGTGAGCAAC CAGCACCCCT GGTTTGGAAT

6241
GGAACAGGAG TATACTCTGA TGGGAACAGA TGGGCACCCT TTTGGTTGGC CTTCCAATGG CTTTCCTGGG CCCCAAGGTC

6321
CGTATTACTG TGGTGTGGGC GCAGACAAAG CCTATGGCAG GGATATCGTG GAGGCTCACT ACCGCGCCTG CTTGTATGCT

6401
GGGGTCAAGA TTACAGGAAC AAATGCTGAG GTCATGCCTG CCCAGTGGGA GTTCCAAATA GGACCCTGTG AAGGAATCCG

6481
CATGGGAGAT CATCTCTGGG TGGCCCGTTT CATCTTGCAT CGAGTATGTG AAGACTTTGG GGTAATAGCA ACCTTTGACC

6561
CCAAGCCCAT TCCTGGGAAC TGGAATGGTG CAGGCTGCCA TACCAACTTT AGCACCAAGG CCATGCGGGA GGAGAATGGT

6641
CTGAAGCACA TCGAGGAGGC CATCGAGAAA CTAAGCAAGC GGCACCGGTA CCACATTCGA GCCTACGATC CCAAGGGGGG

6721
CCTGGACAAT GCCCGTCGTC TGACTGGGTT CCACGAAACG TCCAACATCA ACGACTTTTC TGCTGGTGTC GCCAATCGCA

6801
GTGCCAGCAT CCGCATTCCC CGGACTGTCG GCCAGGAGAA GAAAGGTTAC TTTGAAGACC GCCGCCCCTC TGCCAATTGT

6881
GACCCCTTTG CAGTGACAGA AGCCATCGTC CGCACATGCC TTCTCAATGA GACTGGCGAC GAGCCCTTCC AATACAAAAA

6961
CTAATCTAGA TCCCCCTCGC TTTCTTGCTG TCCAATTTCT ATTAAAGGTT CCTTTGTTCC CTAAGTCCAA CTACTAAACT

7041
GGGGGATATT ATGAAGGGCC TTGAGCATCT GGATTCTGCC TAATAAAAAA CATTTATTTT CATTGCAATG ATGTATTTAA

7121
ATTATTTCTG AATATTTTAC TAAAAAGGGA ATGTGGGAGG TCAGTGCATT TAAAACATAA AGAAATGAAG AGGGGGATCT

7201
TCGCGATACT GCATCGATGA GGGACAGCCC CCCCCCAAAG CCCCCAGGGA TGTAATTACG TCCCTCCCCC GCTAGGGGGC

7281
AGCAGCGAGC CGCCCGGGGC TCCGCTCCGG TCCGGCGCTC CCCCCGCATC CCCGAGCCGG CAGCGTGCGG GGACAGCCCG

7361
GGCACGGGGA AGGTGGCACG GGATCGCTTT CCTCTGAACG CTTCTCGCTG CTCTTTGAGC CTGCAGACAC CTGGGGGGAT

7441
ACGGGGAAAA TAGACACCGC GGTGGAGCTC CAGCTTTTGT TCCCTTTAGT GAGGGTTAAT TAGTTCTTAA TACGACTCAC

7521
TATAGGGCGA ATTGGCTACC GGGCCGCCCA TCGAGGGTAT CATAAGCTTA TATCTATAAC AAGAAAATAT ATATATAATA

7601
AGTTATCACG TAAGTAGAAC ATGAAATAAC AATATAATTA TCGTATGAGT TAAATCTTAA AAGTCACGTA AAAGATAATC

7681
ATGCGTCATT TTGACTCACG CGGTCGTTAT AGTTCAAAAT CAGTGACACT TACCGCATTG ACAAGCACGC CTCACGGGAG

7761
CTCCAAGCGG CGACTGAGAT GTCCTAAATG CACAGCGACG GATTCGCGCT ATTTAGAAAG AGAGAGCAAT ATTTCAAGAA

7841
TGCATGCGTC AATTTTACGC AGACTATCTT TCTAGGGTTA AATCGATAGA TGCGATCCTG CAGGTCTCCC TATAGTGAGT

7921
CGTATTAATT TCGATAAGCC AGCTGCATTA ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT

8001
CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA

8081
CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA

8161
GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC

8241
GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG

8321
CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC

8401
GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT

8481
ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG

8561
TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC

8641
TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT

8721
TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC

8801
GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA

8881
TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG

8961
CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG

9041
GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA

9121
GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG

9201
CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA TCGTGGTGTC ACGCTCGTCG

9281
TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT

9361
TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA

9441
ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT

9521
ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT

9601
CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG

9681
CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA

9761
AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA

9841
TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG

9921
TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT AACCTATAAA AATAGGCGTA TCACGAGGCC CTTTCGTCTC

10001
GCGCGTTTCG GTGATGACGG TGAAAACCTC TGACACATGC AGCTCCCGGA GACGGTCACA GCTTGTCTGT AAGCGGATGC

10081
CGGGAGCAGA CAAGCCCGTC AGGGCGCGTC AGCGGGTGTT GGCGGGTGTC GGGGCTGGCT TAACTATGCG GCATCAGAGC

10161
AGATTGTACT GAGAGTGCAC CATATCGACG CTCTCCCTTA TGCGACTCCT GCATTAGGAA GCAGCCCAGT AGTAGGTTGA

10241
GGCCGTTGAG CACCGCCGCC GCAAGGAATG GTGCATGCAA GGAGATGGCG CCCAACAGTC CCCCGGCCAC GGGGCCTGCC

10321
ACCATACCCA CGCCGAAACA AGCGCTCATG AGCCCGAAGT GGCGAGCCCG ATCTTCCCCA TCGGTGATGT CGGCGATATA

10401
GGCGCCAGCA ACCGCACCTG TGGCGCCGGT GATGCCGGCC ACGATGCGTC CGGCGTAGAG GATCTGGCTA GCGATGACCC

10481
TGCTGATTGG TTCGCTGACC ATTTCCGGGG TGCGGAACGG CGTTACCAGA AACTCAGAAG GTTCGTCCAA CCAAACCGAC

10561
TCTGACGGCA GTTTACGAGA GAGATGATAG GGTCTGCTTC AGTAAGCCAG ATGCTACACA ATTAGGCTTG TACATATTGT

10641
CGTTAGAACG CGGCTACAAT TAATACATAA CCTTATGTAT CATACACATA CGATTTAGGT GACACTATAG AATACACCTG

10721
CAGGACGTCC CAATGATCTT AAGTTAA

pCLD13 (SEQ ID NO: 26):

1
CCCTAGAAAG ATAATCATAT TGTGACGTAC GTTAAAGATA ATCATGCGTA AAATTGACGC ATGTGTTTTA TCGGTCTGTA

81
TATCGAGGTT TATTTATTAA TTTGAATAGA TATTAAGTTT TATTATATTT ACACTTACAT ACTAATAATA AATTCAACAA

161
ACAATTTATT TATGTTTATT TATTTATTAA AAAAAAACAA AAACTCAAAA TTTCTTCTAT AAAGTAACAA AACTTTTATC

241
GAATTTGCAG CCCGGGACTA GCTAGAGGGA CAGCCCCCCC CCAAAGCCCC CAGGGATGTA ATTACGTCCC TCCCCCGCTA

321
GGGGGCAGCA GCGAGCCGCC CGGGGCTCCG CTCCGGTCCG GCGCTCCCCC CGCATCCCCG AGCCGGCAGC GTGCGGGGAC

401
AGCCCGGGCA CGGGGAAGGT GGCACGGGAT CGCTTTCCTC TGAACGCTTC TCGCTGCTCT TTGAGCCTGC AGACACCTGG

481
GGGGATACGG GGAAAACTTA AGATCCGACC GGACGCGTAC TGAGAGCGCT ATTCTGAACT TTTCTTTTGT TCCCTTCCCT

561
TCTACCACAC CCTAATTGTA ATCCATTTTA ATTTCCTGGT CACAGTCCTG TCTCTCCTTC CATTGTACCT TGCCCTTTTC

641
TAAAGAGCGA CTGCAAAGTA TGTTTGCGTA GGTGAGGATC TAAAACTTTA TGAGGTACGA ACATCACAGA ATTACTTTGT

721
AATTTCAGTT TATTGTAGGC TTGGCTTTTT GGGGAGGGTT TACGTCTTAG ACCTCTTAGT GCTTCTTTGT TTCATGGTGT

801
TCTAACTTCG AAGCATCTCT GTAGCTTTAA TGGATTCCTT TTCTGAAAGC TTTGCTCTCT TTCTTCCCCC TCGGCTTTCT

881
CTTAGGCAAG AGGGCTAACT GTAAAGTAAG GCTTACTGCC TTGTGTTTCC AAATGTGTCC GAAGAGGAAG TGTCTTCTGT

961
GAATCCTGTT ATGCATGAAT AACAGGAAAT AGAAAGAAAT TCACTTTCAT TATTATAAAA GTAATATGTT CGTTTAAAAA

1041
ATTCTAATGA AGAGCTGGAG ATGCAACCCA GGGGTAGAGC ACACACTCAG CATGCAGGAG GCCCTGGGTC CAATCTTGGA

1121
ATCTCCTCTC AGTTAACCTG ATCTCTAGCT GATTAGTAGT GAGTGCAAGC CCACTTTCCT CTTCTGCCTC ATTGCTCAGT

1201
GATAACAGCT GTTAAACTTT GTCTTATTCT AAAACTACCT CTGTGCAAAT GCTAGCACAA TAATATATAT CATATGCACA

1281
TGATTTTTTT TTTATCTTGA AAAGTAAGTC AGTATAGCTA CAAAGTTCAC TTGGCATTGT CAACATTTCA CAGGCGTAAT

1361
ATTCCTCCTC TAGTACTGTC CTCTTCATTC TTTGTGACCA AGTTTGGAGA GAGTGCACAA ATGCCAGGGA GGTTTGTGGG

1441
AAGGTTTCTC ATGTTCTGGT AAGGCGAGTA AGAAAATAGT CTCATGCAGG TGAAATGAGT GCTATGCAGT ATATATTATA

1521
CCAGAGAACA GCAAATGACC AAATTCACAC TGAACTAGTT CAGTAAAATT GGCTTTGTCA AAGCTTTCCT TGCTTAAAAT

1601
GTAATTCCCT GTCATCCTAG TTCTGGTCTG GATTCTTTTC CTGGAGTCTT GACTTCCAGA TTCCCTGTGG ACTTTTGTTT

1681
GAGTTTCAAG CTTTTGAAAT ATAGAAACCT ATCTAACTTA ACAAACTTGG GAGAGAAAAG ACTCCAGAAC AACTGAAAAC

1761
AGACCAGGCT AAATGAATAG ACTTTATTCC TCTCTTCTTA CCTGCAGTTT TCAGATATGC AGAGTTGGAG CGGATCTTAG

1841
AGGTTGATTC ATTCATGCCT GAAGAAAACA CATTTTATAG ACCCTGTGCC CAAGTTCGTG GTGGACATCA CCCTTTATTT

1921
ACTAATTGCA CTACATAACA GGCATTTTAG AAGACTGCTC CAGTCAGAGA CCCCGCCTTA GAGGAATCTG TAAACCCTGA

2001
ACTCCTATCA CTCATGAGCA CTAGTTATGT TTGGAATGCC GTATTAAAAC AAAAGTTACA TTTCTAAACT TAAAATTTTC

2081
TAGCACAGAG ACAGTGGGAG TAGCTAACTT TGATAGACAT TTTTCTACTA AAAGTCTTTC TAAGTACATA ATCTTCTGTA

2161
AGTTGGAAAA CAGCAAAATA GAACGTCTCC TACGTAGTTA ATCTTTTTGC ATAATTTGCA CATGTAGGAG TTATTAGTAT

2241
ACGGGTAAGT TTTCACTTTT TCCCCCAACT GGAGTGTCTT GTGGCTGGGT TTGAAAAAGG GAACGGGAGG CCGCTGGAGG

2321
GGATTGGTAA ATGAGATAAA ACACCACTCA TTCAACTCAG TGACTCAGCA TTTAAATTTT CCATAAAAGG ATTAAAGGAA

2401
AATTAAACAA ATTCTTAAAG CCAAGACTCT GGAGAAACTT GTTGGTGTGC TTTAGTTTTC ACTGTTATGA CTCATGAATT

2481
TATGCATAAA TTAGTACATT TATAAAAACA TAGCCTTTTT AGAGTTTTCT GTTTGGCTAA AGTGCCATTG TTAGCATTTG

2561
GAATTACCTT TTTATGTCTT ATATTTTTTC CAAATAAAAA TAAATGTTTC TGCTGTCTTA CTACTGAAAC TACGTTGTGA

2641
GCACTTTAAA TTTCTCAAAG CAGTTTCGCC TGTTATACTT GGCGCTTAGT CATCGTCGTA CACAACAGGA CCTGATTAAG

2721
AAGGCTGTGC TGCCTCTAAG CCGGGCTAGA TTGTAGCCAC TAGCAACCAG GCTGCAATAA TTTCCCTTTG ATGACATCAT

2801
CCACTGTGGA AGAACCCAGT TGCTTCAGCC AGTCGAACTA TACAGTTCCA ACCTCATCAA ATATGGCATC TCCCTTGCCT

2881
GCTATAGCAG GGGGAGGAAA AAATGCCACC ATCTTTTTAA TCTAGCAAGC TTCTCTTTTC TTCATCTTTT TTTTTTTCTT

2961
TTAAAAAAAT TCTGATCATG GATGCTTCTT CCGATCCCTA TTTGCCTTAT GACGGGGGAG GAGACAATAT CCCCTTGAGG

3041
GAATTACATA AAAGAGGTAA GAGCATCCCC TTGCTCTGAA TCCTCTGTTG GTTGTTGTGC ATGCGGCTGG GCGGTTCTGG

3121
GGACAGGCTG TCTGTTGTCC TCTTGCTGCA ATGTGCTGCT TAGTTGCCCT GCCTTGTTGC TGTGGGAGAA TGCGACCTTC

3201
CCAGCAGGGC TGGCCCTCCC TGATTGTTTG CTCTGTGCAG ATTAGCCCTG CTTCAGATCA CATAGGGCTG CAGACTCCAT

3281
CTTCTGTGTG AAAATGCTTT CGGTTTGATT GCAGAAATAA GCTGCCTTTA CAGCCAGCTA AAGTCCTGGT GGTTGGTTGG

3361
CACCTGCAAA GTAGTATTTT TGTACCTCTG GAAACTTATA TTTTCTTTAC ACAGCAATAT CAAGTGCCGG TATGCCATTC

3441
TGTTTTGGCT GCTGCCAATT ACCATGTAGA CTTTGCACCA CAGAGTAATA GTAAAAGCTC CTAGCTGCAT TTTATAACAT

3521
TTAAAAATAG CAGGAAAGAA GAATTATTTT TGATTTAACA TGTTTTTGTC ATTTAACGTC TTAACTGATT GACATACTAT

3601
ATTGTCTGTC TCGTGGGTAT CTTGTACAAC TTGATAGGAT AAAGCAATTT AGTTTTTTTT TTTTTTTTTA AATACATCCA

3681
GAATGTAAGT CGTCAGTAGT TTTCGAACAG ATAAGTAATG GTGTTAATCT TTTGGCAGGC TTTGCCTTGG TCTCCTTAAA

3761
GCTAATTAGG TGTTACTTAA TTAAACTGCT CTTTTGCTCA TTTTCTTAAA TTATTTTTTT AAAAGATAGT TGGCATTTGC

3841
TGTTCTAGAA ATAAACTTCA AGAAACATTC TTTAGCCAGA TGACTTCATG TATGAGCCAT GTTAGTTTGA ATTATTTGCT

3921
TGGTGTTATA AACTTTATGG TTTAATACCA ACTTTTATTA TGTTTACAAG GTAAATAAGG AAAATTTCAA GTACATTTTG

4001
TATCCTGAGA ACAAATTTAA GTTCCATAGA ATTTAGGAAT TACAATGTAT TCAACAGATA CTTACTTGTC ATACTGTGCC

4081
TGCAAAACAA TAATTAGACT CTGAACAGGT GCAACAATTT TCTGTAGAAT TAGACAAGTC TTCTTTTGGC AGGTGTTACT

4161
AAGTAGGCCA TTTCCCAAGG AACAGGGAAT TTGCCAGGCT TTTGTGGTGG AGAGAATAGA ATGAATAAAT GCTGTGGGGA

4241
GTAAAGAGCT TGTCAGAAGA TGATTAGTTC TGTGGCACCA AAACCAAGAG ATCAGTTTTC CTGTGAGAAG TAAAGGAAGC

4321
ATTGTAGAAA AATAGATGTG TTGAAGTCTA CCGGTGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG

4401
ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC

4481
GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT

4561
GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT

4641
CTACGTATTA GTCATCGCTA TTACCATTGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC

4721
GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT

4801
CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCGTTTAGT

4881
GAACCGTCAG ATCTACCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGGTGAGT ACTCCCTCTC AAAAGCGGGC

4961
ATGACTTCTG CGCTAAGATT GTCAGTTTCC AAAAACGAGG AGGATTTGAT ATTCACCTGG CCCGCGGTGA TGCCTTTGAG

5041
GGTGGCCGCG TCCATCTGGT CAGAAAAGAC AATCTTTTTG TTGTCAAGCT TCCTTGATGA TGTCATACTT ATCCTGTCCC

5121
TTTTTTTTCC ACAGCTCGCG GTTGAGGACA AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC

5201
CGAACGGTAC TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCGGATCCGA ATTCATAGAT

5281
AACTGATCCA GTGCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG CCGGTGTGCG TTTGTCTATA TGTTATTTTC

5361
CACCATATTG CCGTCTTTTG GCAATGTGAG GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT

5441
CCCCTCTCGC CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG AAGACAAACA

5521
ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG GTGCCTCTGC GGCCAAAAGC CACGTGTATA

5601
AGATACACCT GCAAAGGCGG CACAACCCCA GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT

5681
CAAGCGTATT CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC TCGGTGCACA

5761
TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AACGTCTAGG CCCCCCGAAC CACGGGGACG TGGTTTTCCT TTGAAAAACA

5841
CGATGATAAA TGGCCACCTC AGCAAGTTCC CACTTGAACA AAAACATCAA GCAAATGTAC TTGTGCCTGC CCCAGGGTGA

5921
GAAAGTCCAA GCCATGTATA TCTGGGTTGA TGGTACTGGA GAAGGACTGC GCTGCAAAAC CCGCACCCTG GACTGTGAGC

6001
CCAAGTGTGT AGAAGAGTTA CCTGAGTGGA ATTTTGATGG CTCTAGTACC TTTCAGTCTG AGGGCTCCAA CAGTGACATG

6081
TATCTCAGCC CTGTTGCCAT GTTTCGGGAC CCCTTCCGCA GAGATCCCAA CAAGCTGGTG TTCTGTGAAG TTTTCAAGTA

6161
CAACCGGAAG CCTGCAGAGA CCAATTTAAG GCACTCGTGT AAACGGATAA TGGACATGGT GAGCAACCAG CACCCCTGGT

6241
TTGGAATGGA ACAGGAGTAT ACTCTGATGG GAACAGATGG GCACCCTTTT GGTTGGCCTT CCAATGGCTT TCCTGGGCCC

6321
CAAGGTCCGT ATTACTGTGG TGTGGGCGCA GACAAAGCCT ATGGCAGGGA TATCGTGGAG GCTCACTACC GCGCCTGCTT

6401
GTATGCTGGG GTCAAGATTA CAGGAACAAA TGCTGAGGTC ATGCCTGCCC AGTGGGAGTT CCAAATAGGA CCCTGTGAAG

6481
GAATCCGCAT GGGAGATCAT CTCTGGGTGG CCCGTTTCAT CTTGCATCGA GTATGTGAAG ACTTTGGGGT AATAGCAACC

6561
TTTGACCCCA AGCCCATTCC TGGGAACTGG AATGGTGCAG GCTGCCATAC CAACTTTAGC ACCAAGGCCA TGCGGGAGGA

6641
GAATGGTCTG AAGCACATCG AGGAGGCCAT CGAGAAACTA AGCAAGCGGC ACCGGTACCA CATTCGAGCC TACGATCCCA

6721
AGGGGGGCCT GGACAATGCC CGTCGTCTGA CTGGGTTCCA CGAAACGTCC AACATCAACG ACTTTTCTGC TGGTGTCGCC

6801
AATCGCAGTG CCAGCATCCG CATTCCCCGG ACTGTCGGCC AGGAGAAGAA AGGTTACTTT GAAGACCGCC GCCCCTCTGC

6881
CAATTGTGAC CCCTTTGCAG TGACAGAAGC CATCGTCCGC ACATGCCTTC TCAATGAGAC TGGCGACGAG CCCTTCCAAT

6961
ACAAAAACTA ATCTAGATCC CCCTCGCTTT CTTGCTGTCC AATTTCTATT AAAGGTTCCT TTGTTCCCTA AGTCCAACTA

7041
CTAAACTGGG GGATATTATG AAGGGCCTTG AGCATCTGGA TTCTGCCTAA TAAAAAACAT TTATTTTCAT TGCAATGATG

7121
TATTTAAATT ATTTCTGAAT ATTTTACTAA AAAGGGAATG TGGGAGGTCA GTGCATTTAA AACATAAAGA AATGAAGAGG

7201
GGGATCTTCG CGATACTGCA TCGATGAGGG ACAGCCCCCC CCCAAAGCCC CCAGGGATGT AATTACGTCC CTCCCCCGCT

7281
AGGGGGCAGC AGCGAGCCGC CCGGGGCTCC GCTCCGGTCC GGCGCTCCCC CCGCATCCCC GAGCCGGCAG CGTGCGGGGA

7361
CAGCCCGGGC ACGGGGAAGG TGGCACGGGA TCGCTTTCCT CTGAACGCTT CTCGCTGCTC TTTGAGCCTG CAGACACCTG

7441
GGGGGATACG GGGAAAATAG ACACCGCGGT GGAGCTCCAG CTTTTGTTCC CTTTAGTGAG GGTTAATTAG TTCTTAATAC

7521
GACTCACTAT AGGGCGAATT GGCTACCGGG CCGCCCATCG AGGGTATCAT AAGCTTATAT CTATAACAAG AAAATATATA

7601
TATAATAAGT TATCACGTAA GTAGAACATG AAATAACAAT ATAATTATCG TATGAGTTAA ATCTTAAAAG TCACGTAAAA

7681
GATAATCATG CGTCATTTTG ACTCACGCGG TCGTTATAGT TCAAAATCAG TGACACTTAC CGCATTGACA AGCACGCCTC

7761
ACGGGAGCTC CAAGCGGCGA CTGAGATGTC CTAAATGCAC AGCGACGGAT TCGCGCTATT TAGAAAGAGA GAGCAATATT

7841
TCAAGAATGC ATGCGTCAAT TTTACGCAGA CTATCTTTCT AGGGTTAAAT CGATAGATGC GATCCTGCAG GTCTCCCTAT

7921
AGTGAGTCGT ATTAATTTCG ATAAGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG

8001
CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC

8081
GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC

8161
GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG

8241
AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC

8321
CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC

8401
TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC

8481
GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG

8561
AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGGACA GTATTTGGTA

8641
TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC

8721
GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG

8801
GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGATCC

8881
TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC

8961
AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT

9041
ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAGCAA

9121
TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC

9201
CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTCACG

9281
CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA

9361
AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA

9441
CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA

9521
ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAAAAG

9601
TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC

9681
ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC

9761
CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTTATC

9841
AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC

9921
CGAAAAGTGC CACCTGACGT CTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTATCA CGAGGCCCTT

10001
TCGTCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCAGC TCCCGGAGAC GGTCACAGCT TGTCTGTAAG

10081
CGGATGCCGG GAGCAGACAA GCCCGTCAGG GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCTGGCTTAA CTATGCGGCA

10161
TCAGAGCAGA TTGTACTGAG AGTGCACCAT ATCGACGCTC TCCCTTATGC GACTCCTGCA TTAGGAAGCA GCCCAGTAGT

10241
AGGTTGAGGC CGTTGAGCAC CGCCGCCGCA AGGAATGGTG CATGCAAGGA GATGGCGCCC AACAGTCCCC CGGCCACGGG

10321
GCCTGCCACC ATACCCACGC CGAAACAAGC GCTCATGAGC CCGAAGTGGC GAGCCCGATC TTCCCCATCG GTGATGTCGG

10401
CGATATAGGC GCCAGCAACC GCACCTGTGG CGCCGGTGAT GCCGGCCACG ATGCGTCCGG CGTAGAGGAT CTGGCTAGCG

10481
ATGACCCTGC TGATTGGTTC GCTGACCATT TCCGGGGTGC GGAACGGCGT TACCAGAAAC TCAGAAGGTT CGTCCAACCA

10561
AACCGACTCT GACGGCAGTT TACGAGAGAG ATGATAGGGT CTGCTTCAGT AAGCCAGATG CTACACAATT AGGCTTGTAC

10641
ATATTGTCGT TAGAACGCGG CTACAATTAA TACATAACCT TATGTATCAT ACACATACGA TTTAGGTGAC ACTATAGAAT

10721
ACACCTGCAG GACGTCCCAA TGATCTTAAG TTAA

pCLD15 (SEQ ID NO: 27):

1
CCCTAGAAAG ATAATCATAT TGTGACGTAC GTTAAAGATA ATCATGCGTA AAATTGACGC ATGTGTTTTA TCGGTCTGTA

81
TATCGAGGTT TATTTATTAA TTTGAATAGA TATTAAGTTT TATTATATTT ACACTTACAT ACTAATAATA AATTCAACAA

161
ACAATTTATT TATGTTTATT TATTTATTAA AAAAAAACAA AAACTCAAAA TTTCTTCTAT AAAGTAACAA AACTTTTATC

241
GAATTTGCAG CCCGGGACTA GCTAGAGGGA CAGCCCCCCC CCAAAGCCCC CAGGGATGTA ATTACGTCCC TCCCCCGCTA

321
GGGGGCAGCA GCGAGCCGCC CGGGGCTCCG CTCCGGTCCG GCGCTCCCCC CGCATCCCCG AGCCGGCAGC GTGCGGGGAC

401
AGCCCGGGCA CGGGGAAGGT GGCACGGGAT CGCTTTCCTC TGAACGCTTC TCGCTGCTCT TTGAGCCTGC AGACACCTGG

481
GGGGATACGG GGAAAACTTA AGATCCGACC GGACGCGTAC TGAGAGCGCT ATTCTGAACT TTTCTTTTGT TCCCTTCCCT

561
TCTACCACAC CCTAATTGTA ATCCATTTTA ATTTCCTGGT CACAGTCCTG TCTCTCCTTC CATTGTACCT TGCCCTTTTC

641
TAAAGAGCGA CTGCAAAGTA TGTTTGCGTA GGTGAGGATC TAAAACTTTA TGAGGTACGA ACATCACAGA ATTACTTTGT

721
AATTTCAGTT TATTGTAGGC TTGGCTTTTT GGGGAGGGTT TACGTCTTAG ACCTCTTAGT GCTTCTTTGT TTCATGGTGT

801
TCTAACTTCG AAGCATCTCT GTAGCTTTAA TGGATTCCTT TTCTGAAAGC TTTGCTCTCT TTCTTCCCCC TCGGCTTTCT

881
CTTAGGCAAG AGGGCTAACT GTAAAGTAAG GCTTACTGCC TTGTGTTTCC AAATGTGTCC GAAGAGGAAG TGTCTTCTGT

961
GAATCCTGTT ATGCATGAAT AACAGGAAAT AGAAAGAAAT TCACTTTCAT TATTATAAAA GTAATATGTT CGTTTAAAAA

1041
ATTCTAATGA AGAGCTGGAG ATGCAACCCA GGGGTAGAGC ACACACTCAG CATGCAGGAG GCCCTGGGTC CAATCTTGGA

1121
ATCTCCTCTC AGTTAACCTG ATCTCTAGCT GATTAGTAGT GAGTGCAAGC CCACTTTCCT CTTCTGCCTC ATTGCTCAGT

1201
GATAACAGCT GTTAAACTTT GTCTTATTCT AAAACTACCT CTGTGCAAAT GCTAGCACAA TAATATATAT CATATGCACA

1281
TGATTTTTTT TTTATCTTGA AAAGTAAGTC AGTATAGCTA CAAAGTTCAC TTGGCATTGT CAACATTTCA CAGGCGTAAT

1361
ATTCCTCCTC TAGTACTGTC CTCTTCATTC TTTGTGACCA AGTTTGGAGA GAGTGCACAA ATGCCAGGGA GGTTTGTGGG

1441
AAGGTTTCTC ATGTTCTGGT AAGGCGAGTA AGAAAATAGT CTCATGCAGG TGAAATGAGT GCTATGCAGT ATATATTATA

1521
CCAGAGAACA GCAAATGACC AAATTCACAC TGAACTAGTT CAGTAAAATT GGCTTTGTCA AAGCTTTCCT TGCTTAAAAT

1601
GTAATTCCCT GTCATCCTAG TTCTGGTCTG GATTCTTTTC CTGGAGTCTT GACTTCCAGA TTCCCTGTGG ACTTTTGTTT

1681
GAGTTTCAAG CTTTTGAAAT ATAGAAACCT ATCTAACTTA ACAAACTTGG GAGAGAAAAG ACTCCAGAAC AACTGAAAAC

1761
AGACCAGGCT AAATGAATAG ACTTTATTCC TCTCTTCTTA CCTGCAGTTT TCAGATATGC AGAGTTGGAG CGGATCTTAG

1841
AGGTTGATTC ATTCATGCCT GAAGAAAACA CATTTTATAG ACCCTGTGCC CAAGTTCGTG GTGGACATCA CCCTTTATTT

1921
ACTAATTGCA CTACATAACA GGCATTTTAG AAGACTGCTC CAGTCAGAGA CCCCGCCTTA GAGGAATCTG TAAACCCTGA

2001
ACTCCTATCA CTCATGAGCA CTAGTTATGT TTGGAATGCC GTATTAAAAC AAAAGTTACA TTTCTAAACT TAAAATTTTC

2081
TAGCACAGAG ACAGTGGGAG TAGCTAACTT TGATAGACAT TTTTCTACTA AAAGTCTTTC TAAGTACATA ATCTTCTGTA

2161
AGTTGGAAAA CAGCAAAATA GAACGTCTCC TACGTAGTTA ATCTTTTTGC ATAATTTGCA CATGTAGGAG TTATTAGTAT

2241
ACGGGTAAGT TTTCACTTTT TCCCCCAACT GGAGTGTCTT GTGGCTGGGT TTGAAAAAGG GAACGGGAGG CCGCTGGAGG

2321
GGATTGGTAA ATGAGATAAA ACACCACTCA TTCAACTCAG TGACTCAGCA TTTAAATTTT CCATAAAAGG ATTAAAGGAA

2401
AATTAAACAA ATTCTTAAAG CCAAGACTCT GGAGAAACTT GTTGGTGTGC TTTAGTTTTC ACTGTTATGA CTCATGAATT

2481
TATGCATAAA TTAGTACATT TATAAAAACA TAGCCTTTTT AGAGTTTTCT GTTTGGCTAA AGTGCCATTG TTAGCATTTG

2561
GAATTACCTT TTTATGTCTT ATATTTTTTC CAAATAAAAA TAAATGTTTC TGCTGTCTTA CTACTGAAAC TACGTTGTGA

2641
GCACTTTAAA TTTCTCAAAG CAGTTTCGCC TGTTATACTT GGCGCTTAGT CATCGTCGTA CACAACAGGA CCTGATTAAG

2721
AAGGCTGTGC TGCCTCTAAG CCGGGCTAGA TTGTAGCCAC TAGCAACCAG GCTGCAATAA TTTCCCTTTG ATGACATCAT

2801
CCACTGTGGA AGAACCCAGT TGCTTCAGCC AGTCGAACTA TACAGTTCCA ACCTCATCAA ATATGGCATC TCCCTTGCCT

2881
GCTATAGCAG GGGGAGGAAA AAATGCCACC ATCTTTTTAA TCTAGCAAGC TTCTCTTTTC TTCATCTTTT TTTTTTTCTT

2961
TTAAAAAAAT TCTGATCATG GATGCTTCTT CCGATCCCTA TTTGCCTTAT GACGGGGGAG GAGACAATAT CCCCTTGAGG

3041
GAATTACATA AAAGAGGTAA GAGCATCCCC TTGCTCTGAA TCCTCTGTTG GTTGTTGTGC ATGCGGCTGG GCGGTTCTGG

3121
GGACAGGCTG TCTGTTGTCC TCTTGCTGCA ATGTGCTGCT TAGTTGCCCT GCCTTGTTGC TGTGGGAGAA TGCGACCTTC

3201
CCAGCAGGGC TGGCCCTCCC TGATTGTTTG CTCTGTGCAG ATTAGCCCTG CTTCAGATCA CATAGGGCTG CAGACTCCAT

3281
CTTCTGTGTG AAAATGCTTT CGGTTTGATT GCAGAAATAA GCTGCCTTTA CAGCCAGCTA AAGTCCTGGT GGTTGGTTGG

3361
CACCTGCAAA GTAGTATTTT TGTACCTCTG GAAACTTATA TTTTCTTTAC ACAGCAATAT CAAGTGCCGG TATGCCATTC

3441
TGTTTTGGCT GCTGCCAATT ACCATGTAGA CTTTGCACCA CAGAGTAATA GTAAAAGCTC CTAGCTGCAT TTTATAACAT

3521
TTAAAAATAG CAGGAAAGAA GAATTATTTT TGATTTAACA TGTTTTTGTC ATTTAACGTC TTAACTGATT GACATACTAT

3601
ATTGTCTGTC TCGTGGGTAT CTTGTACAAC TTGATAGGAT AAAGCAATTT AGTTTTTTTT TTTTTTTTTA AATACATCCA

3681
GAATGTAAGT CGTCAGTAGT TTTCGAACAG ATAAGTAATG GTGTTAATCT TTTGGCAGGC TTTGCCTTGG TCTCCTTAAA

3761
GCTAATTAGG TGTTACTTAA TTAAACTGCT CTTTTGCTCA TTTTCTTAAA TTATTTTTTT AAAAGATAGT TGGCATTTGC

3841
TGTTCTAGAA ATAAACTTCA AGAAACATTC TTTAGCCAGA TGACTTCATG TATGAGCCAT GTTAGTTTGA ATTATTTGCT

3921
TGGTGTTATA AACTTTATGG TTTAATACCA ACTTTTATTA TGTTTACAAG GTAAATAAGG AAAATTTCAA GTACATTTTG

4001
TATCCTGAGA ACAAATTTAA GTTCCATAGA ATTTAGGAAT TACAATGTAT TCAACAGATA CTTACTTGTC ATACTGTGCC

4081
TGCAAAACAA TAATTAGACT CTGAACAGGT GCAACAATTT TCTGTAGAAT TAGACAAGTC TTCTTTTGGC AGGTGTTACT

4161
AAGTAGGCCA TTTCCCAAGG AACAGGGAAT TTGCCAGGCT TTTGTGGTGG AGAGAATAGA ATGAATAAAT GCTGTGGGGA

4241
GTAAAGAGCT TGTCAGAAGA TGATTAGTTC TGTGGCACCA AAACCAAGAG ATCAGTTTTC CTGTGAGAAG TAAAGGAAGC

4321
ATTGTAGAAA AATAGATGTG TTGAAGTCTA CCGGTGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG

4401
ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC

4481
GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT

4561
GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT

4641
CTACGTATTA GTCATCGCTA TTACCATTGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC

4721
GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT

4801
CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCGTTTAGT

4881
GAACCGTCAG ATCTACCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGGTGAGT ACTCCCTCTC AAAAGCGGGC

4961
ATGACTTCTG CGCTAAGATT GTCAGTTTCC AAAAACGAGG AGGATTTGAT ATTCACCTGG CCCGCGGTGA TGCCTTTGAG

5041
GGTGGCCGCG TCCATCTGGT CAGAAAAGAC AATCTTTTTG TTGTCAAGCT TCCTTGATGA TGTCATACTT ATCCTGTCCC

5121
TTTTTTTTCC ACAGCTCGCG GTTGAGGACA AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC

5201
CGAACGGTAC TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCGGATCCGA ATTCATAGAT

5281
AACTGATCCA GTGCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG CCGGTGTGCG TTTGTCTATA TGTTATTTTC

5361
CACCATATTG CCGTCTTTTG GCAATGTGAG GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT

5441
CCCCTCTCGC CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG AAGACAAACA

5521
ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG GTGCCTCTGC GGCCAAAAGC CACGTGTATA

5601
AGATACACCT GCAAAGGCGG CACAACCCCA GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT

5681
CAAGCGTATT CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC TCGGTGCACA

5761
TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA CCACGGGGAC GTGGTTTTCC TTTGAAAAAC

5841
ACGATGATAA TATGGCCACA ACCATGGCCA CCTCAGCAAG TTCCCACTTG AACAAAAACA TCAAGCAAAT GTACTTGTGC

5921
CTGCCCCAGG GTGAGAAAGT CCAAGCCATG TATATCTGGG TTGATGGTAC TGGAGAAGGA CTGCGCTGCA AAACCCGCAC

6001
CCTGGACTGT GAGCCCAAGT GTGTAGAAGA GTTACCTGAG TGGAATTTTG ATGGCTCTAG TACCTTTCAG TCTGAGGGCT

6081
CCAACAGTGA CATGTATCTC AGCCCTGTTG CCATGTTTCG GGACCCCTTC CGCAGAGATC CCAACAAGCT GGTGTTCTGT

6161
GAAGTTTTCA AGTACAACCG GAAGCCTGCA GAGACCAATT TAAGGCACTC GTGTAAACGG ATAATGGACA TGGTGAGCAA

6241
CCAGCACCCC TGGTTTGGAA TGGAACAGGA GTATACTCTG ATGGGAACAG ATGGGCACCC TTTTGGTTGG CCTTCCAATG

6321
GCTTTCCTGG GCCCCAAGGT CCGTATTACT GTGGTGTGGG CGCAGACAAA GCCTATGGCA GGGATATCGT GGAGGCTCAC

6401
TACCGCGCCT GCTTGTATGC TGGGGTCAAG ATTACAGGAA CAAATGCTGA GGTCATGCCT GCCCAGTGGG AGTTCCAAAT

6481
AGGACCCTGT GAAGGAATCC GCATGGGAGA TCATCTCTGG GTGGCCCGTT TCATCTTGCA TCGAGTATGT GAAGACTTTG

6561
GGGTAATAGC AACCTTTGAC CCCAAGCCCA TTCCTGGGAA CTGGAATGGT GCAGGCTGCC ATACCAACTT TAGCACCAAG

6641
GCCATGCGGG AGGAGAATGG TCTGAAGCAC ATCGAGGAGG CCATCGAGAA ACTAAGCAAG CGGCACCGGT ACCACATTCG

6721
AGCCTACGAT CCCAAGGGGG GCCTGGACAA TGCCCGTCGT CTGACTGGGT TCCACGAAAC GTCCAACATC AACGACTTTT

6801
CTGCTGGTGT CGCCAATCGC AGTGCCAGCA TCCGCATTCC CCGGACTGTC GGCCAGGAGA AGAAAGGTTA CTTTGAAGAC

6881
CGCCGCCCCT CTGCCAATTG TGACCCCTTT GCAGTGACAG AAGCCATCGT CCGCACATGC CTTCTCAATG AGACTGGCGA

6961
CGAGCCCTTC CAATACAAAA ACTAATCTAG ATCCCCCTCG CTTTCTTGCT GTCCAATTTC TATTAAAGGT TCCTTTGTTC

7041
CCTAAGTCCA ACTACTAAAC TGGGGGATAT TATGAAGGGC CTTGAGCATC TGGATTCTGC CTAATAAAAA ACATTTATTT

7121
TCATTGCAAT GATGTATTTA AATTATTTCT GAATATTTTA CTAAAAAGGG AATGTGGGAG GTCAGTGCAT TTAAAACATA

7201
AAGAAATGAA GAGGGGGATC TTCGCGATAC TGCATCGATG AGGGACAGCC CCCCCCCAAA GCCCCCAGGG ATGTAATTAC

7281
GTCCCTCCCC CGCTAGGGGG CAGCAGCGAG CCGCCCGGGG CTCCGCTCCG GTCCGGCGCT CCCCCCGCAT CCCCGAGCCG

7361
GCAGCGTGCG GGGACAGCCC GGGCACGGGG AAGGTGGCAC GGGATCGCTT TCCTCTGAAC GCTTCTCGCT GCTCTTTGAG

7441
CCTGCAGACA CCTGGGGGGA TACGGGGAAA ATAGACACCG CGGTGGAGCT CCAGCTTTTG TTCCCTTTAG TGAGGGTTAA

7521
TTAGTTCTTA ATACGACTCA CTATAGGGCG AATTGGCTAC CGGGCCGCCC ATCGAGGGTA TCATAAGCTT ATATCTATAA

7601
CAAGAAAATA TATATATAAT AAGTTATCAC GTAAGTAGAA CATGAAATAA CAATATAATT ATCGTATGAG TTAAATCTTA

7681
AAAGTCACGT AAAAGATAAT CATGCGTCAT TTTGACTCAC GCGGTCGTTA TAGTTCAAAA TCAGTGACAC TTACCGCATT

7761
GACAAGCACG CCTCACGGGA GCTCCAAGCG GCGACTGAGA TGTCCTAAAT GCACAGCGAC GGATTCGCGC TATTTAGAAA

7841
GAGAGAGCAA TATTTCAAGA ATGCATGCGT CAATTTTACG CAGACTATCT TTCTAGGGTT AAATCGATAG ATGCGATCCT

7921
GCAGGTCTCC CTATAGTGAG TCGTATTAAT TTCGATAAGC CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG

8001
GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA

8081
GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA

8161
AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC

8241
GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC

8321
TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC

8401
ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC

8481
GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT

8561
AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG

8641
GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

8721
CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG

8801
ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT

8881
CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

8961
ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG

9041
TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC

9121
AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

9201
CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC

9281
ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC

9361
CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

9441
TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC

9521
AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG

9601
CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

9681
GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA

9761
GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA

9841
TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

9921
CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT TAACCTATAA AAATAGGCGT

10001
ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC

10081
AGCTTGTCTG TAAGCGGATG CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC

10161
TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATCGAC GCTCTCCCTT ATGCGACTCC TGCATTAGGA

10241
AGCAGCCCAG TAGTAGGTTG AGGCCGTTGA GCACCGCCGC CGCAAGGAAT GGTGCATGCA AGGAGATGGC GCCCAACAGT

10321
CCCCCGGCCA CGGGGCCTGC CACCATACCC ACGCCGAAAC AAGCGCTCAT GAGCCCGAAG TGGCGAGCCC GATCTTCCCC

10401
ATCGGTGATG TCGGCGATAT AGGCGCCAGC AACCGCACCT GTGGCGCCGG TGATGCCGGC CACGATGCGT CCGGCGTAGA

10481
GGATCTGGCT AGCGATGACC CTGCTGATTG GTTCGCTGAC CATTTCCGGG GTGCGGAACG GCGTTACCAG AAACTCAGAA

10561
GGTTCGTCCA ACCAAACCGA CTCTGACGGC AGTTTACGAG AGAGATGATA GGGTCTGCTT CAGTAAGCCA GATGCTACAC

10641
AATTAGGCTT GTACATATTG TCGTTAGAAC GCGGCTACAA TTAATACATA ACCTTATGTA TCATACACAT ACGATTTAGG

10721
TGACACTATA GAATACACCT GCAGGACGTC CCAATGATCT TAAGTTAA

pCLD16 (SEQ ID NO: 28):

1
CCCTAGAAAG ATAATCATAT TGTGACGTAC GTTAAAGATA ATCATGCGTA AAATTGACGC ATGTGTTTTA TCGGTCTGTA

81
TATCGAGGTT TATTTATTAA TTTGAATAGA TATTAAGTTT TATTATATTT ACACTTACAT ACTAATAATA AATTCAACAA

161
ACAATTTATT TATGTTTATT TATTTATTAA AAAAAAACAA AAACTCAAAA TTTCTTCTAT AAAGTAACAA AACTTTTATC

241
GAATTTGCAG CCCGGGACTA GCTAGAGGGA CAGCCCCCCC CCAAAGCCCC CAGGGATGTA ATTACGTCCC TCCCCCGCTA

321
GGGGGCAGCA GCGAGCCGCC CGGGGCTCCG CTCCGGTCCG GCGCTCCCCC CGCATCCCCG AGCCGGCAGC GTGCGGGGAC

401
AGCCCGGGCA CGGGGAAGGT GGCACGGGAT CGCTTTCCTC TGAACGCTTC TCGCTGCTCT TTGAGCCTGC AGACACCTGG

481
GGGGATACGG GGAAAACTTA AGATCCGACC GGACGCGTAC TGAGAGCGCT ATTCTGAACT TTTCTTTTGT TCCCTTCCCT

561
TCTACCACAC CCTAATTGTA ATCCATTTTA ATTTCCTGGT CACAGTCCTG TCTCTCCTTC CATTGTACCT TGCCCTTTTC

641
TAAAGAGCGA CTGCAAAGTA TGTTTGCGTA GGTGAGGATC TAAAACTTTA TGAGGTACGA ACATCACAGA ATTACTTTGT

721
AATTTCAGTT TATTGTAGGC TTGGCTTTTT GGGGAGGGTT TACGTCTTAG ACCTCTTAGT GCTTCTTTGT TTCATGGTGT

801
TCTAACTTCG AAGCATCTCT GTAGCTTTAA TGGATTCCTT TTCTGAAAGC TTTGCTCTCT TTCTTCCCCC TCGGCTTTCT

881
CTTAGGCAAG AGGGCTAACT GTAAAGTAAG GCTTACTGCC TTGTGTTTCC AAATGTGTCC GAAGAGGAAG TGTCTTCTGT

961
GAATCCTGTT ATGCATGAAT AACAGGAAAT AGAAAGAAAT TCACTTTCAT TATTATAAAA GTAATATGTT CGTTTAAAAA

1041
ATTCTAATGA AGAGCTGGAG ATGCAACCCA GGGGTAGAGC ACACACTCAG CATGCAGGAG GCCCTGGGTC CAATCTTGGA

1121
ATCTCCTCTC AGTTAACCTG ATCTCTAGCT GATTAGTAGT GAGTGCAAGC CCACTTTCCT CTTCTGCCTC ATTGCTCAGT

1201
GATAACAGCT GTTAAACTTT GTCTTATTCT AAAACTACCT CTGTGCAAAT GCTAGCACAA TAATATATAT CATATGCACA

1281
TGATTTTTTT TTTATCTTGA AAAGTAAGTC AGTATAGCTA CAAAGTTCAC TTGGCATTGT CAACATTTCA CAGGCGTAAT

1361
ATTCCTCCTC TAGTACTGTC CTCTTCATTC TTTGTGACCA AGTTTGGAGA GAGTGCACAA ATGCCAGGGA GGTTTGTGGG

1441
AAGGTTTCTC ATGTTCTGGT AAGGCGAGTA AGAAAATAGT CTCATGCAGG TGAAATGAGT GCTATGCAGT ATATATTATA

1521
CCAGAGAACA GCAAATGACC AAATTCACAC TGAACTAGTT CAGTAAAATT GGCTTTGTCA AAGCTTTCCT TGCTTAAAAT

1601
GTAATTCCCT GTCATCCTAG TTCTGGTCTG GATTCTTTTC CTGGAGTCTT GACTTCCAGA TTCCCTGTGG ACTTTTGTTT

1681
GAGTTTCAAG CTTTTGAAAT ATAGAAACCT ATCTAACTTA ACAAACTTGG GAGAGAAAAG ACTCCAGAAC AACTGAAAAC

1761
AGACCAGGCT AAATGAATAG ACTTTATTCC TCTCTTCTTA CCTGCAGTTT TCAGATATGC AGAGTTGGAG CGGATCTTAG

1841
AGGTTGATTC ATTCATGCCT GAAGAAAACA CATTTTATAG ACCCTGTGCC CAAGTTCGTG GTGGACATCA CCCTTTATTT

1921
ACTAATTGCA CTACATAACA GGCATTTTAG AAGACTGCTC CAGTCAGAGA CCCCGCCTTA GAGGAATCTG TAAACCCTGA

2001
ACTCCTATCA CTCATGAGCA CTAGTTATGT TTGGAATGCC GTATTAAAAC AAAAGTTACA TTTCTAAACT TAAAATTTTC

2081
TAGCACAGAG ACAGTGGGAG TAGCTAACTT TGATAGACAT TTTTCTACTA AAAGTCTTTC TAAGTACATA ATCTTCTGTA

2161
AGTTGGAAAA CAGCAAAATA GAACGTCTCC TACGTAGTTA ATCTTTTTGC ATAATTTGCA CATGTAGGAG TTATTAGTAT

2241
ACGGGTAAGT TTTCACTTTT TCCCCCAACT GGAGTGTCTT GTGGCTGGGT TTGAAAAAGG GAACGGGAGG CCGCTGGAGG

2321
GGATTGGTAA ATGAGATAAA ACACCACTCA TTCAACTCAG TGACTCAGCA TTTAAATTTT CCATAAAAGG ATTAAAGGAA

2401
AATTAAACAA ATTCTTAAAG CCAAGACTCT GGAGAAACTT GTTGGTGTGC TTTAGTTTTC ACTGTTATGA CTCATGAATT

2481
TATGCATAAA TTAGTACATT TATAAAAACA TAGCCTTTTT AGAGTTTTCT GTTTGGCTAA AGTGCCATTG TTAGCATTTG

2561
GAATTACCTT TTTATGTCTT ATATTTTTTC CAAATAAAAA TAAATGTTTC TGCTGTCTTA CTACTGAAAC TACGTTGTGA

2641
GCACTTTAAA TTTCTCAAAG CAGTTTCGCC TGTTATACTT GGCGCTTAGT CATCGTCGTA CACAACAGGA CCTGATTAAG

2721
AAGGCTGTGC TGCCTCTAAG CCGGGCTAGA TTGTAGCCAC TAGCAACCAG GCTGCAATAA TTTCCCTTTG ATGACATCAT

2801
CCACTGTGGA AGAACCCAGT TGCTTCAGCC AGTCGAACTA TACAGTTCCA ACCTCATCAA ATATGGCATC TCCCTTGCCT

2881
GCTATAGCAG GGGGAGGAAA AAATGCCACC ATCTTTTTAA TCTAGCAAGC TTCTCTTTTC TTCATCTTTT TTTTTTTCTT

2961
TTAAAAAAAT TCTGATCATG GATGCTTCTT CCGATCCCTA TTTGCCTTAT GACGGGGGAG GAGACAATAT CCCCTTGAGG

3041
GAATTACATA AAAGAGGTAA GAGCATCCCC TTGCTCTGAA TCCTCTGTTG GTTGTTGTGC ATGCGGCTGG GCGGTTCTGG

3121
GGACAGGCTG TCTGTTGTCC TCTTGCTGCA ATGTGCTGCT TAGTTGCCCT GCCTTGTTGC TGTGGGAGAA TGCGACCTTC

3201
CCAGCAGGGC TGGCCCTCCC TGATTGTTTG CTCTGTGCAG ATTAGCCCTG CTTCAGATCA CATAGGGCTG CAGACTCCAT

3281
CTTCTGTGTG AAAATGCTTT CGGTTTGATT GCAGAAATAA GCTGCCTTTA CAGCCAGCTA AAGTCCTGGT GGTTGGTTGG

3361
CACCTGCAAA GTAGTATTTT TGTACCTCTG GAAACTTATA TTTTCTTTAC ACAGCAATAT CAAGTGCCGG TATGCCATTC

3441
TGTTTTGGCT GCTGCCAATT ACCATGTAGA CTTTGCACCA CAGAGTAATA GTAAAAGCTC CTAGCTGCAT TTTATAACAT

3521
TTAAAAATAG CAGGAAAGAA GAATTATTTT TGATTTAACA TGTTTTTGTC ATTTAACGTC TTAACTGATT GACATACTAT

3601
ATTGTCTGTC TCGTGGGTAT CTTGTACAAC TTGATAGGAT AAAGCAATTT AGTTTTTTTT TTTTTTTTTA AATACATCCA

3681
GAATGTAAGT CGTCAGTAGT TTTCGAACAG ATAAGTAATG GTGTTAATCT TTTGGCAGGC TTTGCCTTGG TCTCCTTAAA

3761
GCTAATTAGG TGTTACTTAA TTAAACTGCT CTTTTGCTCA TTTTCTTAAA TTATTTTTTT AAAAGATAGT TGGCATTTGC

3841
TGTTCTAGAA ATAAACTTCA AGAAACATTC TTTAGCCAGA TGACTTCATG TATGAGCCAT GTTAGTTTGA ATTATTTGCT

3921
TGGTGTTATA AACTTTATGG TTTAATACCA ACTTTTATTA TGTTTACAAG GTAAATAAGG AAAATTTCAA GTACATTTTG

4001
TATCCTGAGA ACAAATTTAA GTTCCATAGA ATTTAGGAAT TACAATGTAT TCAACAGATA CTTACTTGTC ATACTGTGCC

4081
TGCAAAACAA TAATTAGACT CTGAACAGGT GCAACAATTT TCTGTAGAAT TAGACAAGTC TTCTTTTGGC AGGTGTTACT

4161
AAGTAGGCCA TTTCCCAAGG AACAGGGAAT TTGCCAGGCT TTTGTGGTGG AGAGAATAGA ATGAATAAAT GCTGTGGGGA

4241
GTAAAGAGCT TGTCAGAAGA TGATTAGTTC TGTGGCACCA AAACCAAGAG ATCAGTTTTC CTGTGAGAAG TAAAGGAAGC

4321
ATTGTAGAAA AATAGATGTG TTGAAGTCTA CCGGTGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG

4401
ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC

4481
GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT

4561
GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT

4641
CTACGTATTA GTCATCGCTA TTACCATTGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC

4721
GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT

4801
CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCGTTTAGT

4881
GAACCGTCAG ATCTACCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGGTGAGT ACTCCCTCTC AAAAGCGGGC

4961
ATGACTTCTG CGCTAAGATT GTCAGTTTCC AAAAACGAGG AGGATTTGAT ATTCACCTGG CCCGCGGTGA TGCCTTTGAG

5041
GGTGGCCGCG TCCATCTGGT CAGAAAAGAC AATCTTTTTG TTGTCAAGCT TCCTTGATGA TGTCATACTT ATCCTGTCCC

5121
TTTTTTTTCC ACAGCTCGCG GTTGAGGACA AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC

5201
CGAACGGTAC TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCGGATCCGA ATTCATAGAT

5281
AACTGATCCA GTGCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG CCGGTGTGCG TTTGTCTATA TGTTATTTTC

5361
CACCATATTG CCGTCTTTTG GCAATGTGAG GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT

5441
CCCCTCTCGC CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG AAGACAAACA

5521
ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG GTGCCTCTGC GGCCAAAAGC CACGTGTATA

5601
AGATACACCT GCAAAGGCGG CACAACCCCA GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT

5681
CAAGCGTATT CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC TCGGTGCACA

5761
TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA CCACGGGGAC GTGGTTTTCC TTTGAAAAAC

5841
ACGATGATAA ATGGCCACCT CAGCAAGTTC CCACTTGAAC AAAAACATCA AGCAAATGTA CTTGTGCCTG CCCCAGGGTG

5921
AGAAAGTCCA AGCCATGTAT ATCTGGGTTG ATGGTACTGG AGAAGGACTG CGCTGCAAAA CCCGCACCCT GGACTGTGAG

6001
CCCAAGTGTG TAGAAGAGTT ACCTGAGTGG AATTTTGATG GCTCTAGTAC CTTTCAGTCT GAGGGCTCCA ACAGTGACAT

6081
GTATCTCAGC CCTGTTGCCA TGTTTCGGGA CCCCTTCCGC AGAGATCCCA ACAAGCTGGT GTTCTGTGAA GTTTTCAAGT

6161
ACAACCGGAA GCCTGCAGAG ACCAATTTAA GGCACTCGTG TAAACGGATA ATGGACATGG TGAGCAACCA GCACCCCTGG

6241
TTTGGAATGG AACAGGAGTA TACTCTGATG GGAACAGATG GGCACCCTTT TGGTTGGCCT TCCAATGGCT TTCCTGGGCC

6321
CCAAGGTCCG TATTACTGTG GTGTGGGCGC AGACAAAGCC TATGGCAGGG ATATCGTGGA GGCTCACTAC CGCGCCTGCT

6401
TGTATGCTGG GGTCAAGATT ACAGGAACAA ATGCTGAGGT CATGCCTGCC CAGTGGGAGT TCCAAATAGG ACCCTGTGAA

6481
GGAATCCGCA TGGGAGATCA TCTCTGGGTG GCCCGTTTCA TCTTGCATCG AGTATGTGAA GACTTTGGGG TAATAGCAAC

6561
CTTTGACCCC AAGCCCATTC CTGGGAACTG GAATGGTGCA GGCTGCCATA CCAACTTTAG CACCAAGGCC ATGCGGGAGG

6641
AGAATGGTCT GAAGCACATC GAGGAGGCCA TCGAGAAACT AAGCAAGCGG CACCGGTACC ACATTCGAGC CTACGATCCC

6721
AAGGGGGGCC TGGACAATGC CCGTCGTCTG ACTGGGTTCC ACGAAACGTC CAACATCAAC GACTTTTCTG CTGGTGTCGC

6801
CAATCGCAGT GCCAGCATCC GCATTCCCCG GACTGTCGGC CAGGAGAAGA AAGGTTACTT TGAAGACCGC CGCCCCTCTG

6881
CCAATTGTGA CCCCTTTGCA GTGACAGAAG CCATCGTCCG CACATGCCTT CTCAATGAGA CTGGCGACGA GCCCTTCCAA

6961
TACAAAAACT AATCTAGATC CCCCTCGCTT TCTTGCTGTC CAATTTCTAT TAAAGGTTCC TTTGTTCCCT AAGTCCAACT

7041
ACTAAACTGG GGGATATTAT GAAGGGCCTT GAGCATCTGG ATTCTGCCTA ATAAAAAACA TTTATTTTCA TTGCAATGAT

7121
GTATTTAAAT TATTTCTGAA TATTTTACTA AAAAGGGAAT GTGGGAGGTC AGTGCATTTA AAACATAAAG AAATGAAGAG

7201
GGGGATCTTC GCGATACTGC ATCGATGAGG GACAGCCCCC CCCCAAAGCC CCCAGGGATG TAATTACGTC CCTCCCCCGC

7281
TAGGGGGCAG CAGCGAGCCG CCCGGGGCTC CGCTCCGGTC CGGCGCTCCC CCCGCATCCC CGAGCCGGCA GCGTGCGGGG

7361
ACAGCCCGGG CACGGGGAAG GTGGCACGGG ATCGCTTTCC TCTGAACGCT TCTCGCTGCT CTTTGAGCCT GCAGACACCT

7441
GGGGGGATAC GGGGAAAATA GACACCGCGG TGGAGCTCCA GCTTTTGTTC CCTTTAGTGA GGGTTAATTA GTTCTTAATA

7521
CGACTCACTA TAGGGCGAAT TGGCTACCGG GCCGCCCATC GAGGGTATCA TAAGCTTATA TCTATAACAA GAAAATATAT

7601
ATATAATAAG TTATCACGTA AGTAGAACAT GAAATAACAA TATAATTATC GTATGAGTTA AATCTTAAAA GTCACGTAAA

7681
AGATAATCAT GCGTCATTTT GACTCACGCG GTCGTTATAG TTCAAAATCA GTGACACTTA CCGCATTGAC AAGCACGCCT

7761
CACGGGAGCT CCAAGCGGCG ACTGAGATGT CCTAAATGCA CAGCGACGGA TTCGCGCTAT TTAGAAAGAG AGAGCAATAT

7841
TTCAAGAATG CATGCGTCAA TTTTACGCAG ACTATCTTTC TAGGGTTAAA TCGATAGATG CGATCCTGCA GGTCTCCCTA

7921
TAGTGAGTCG TATTAATTTC GATAAGCCAG CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG

8001
GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG

8081
CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC

8161
CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA

8241
GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA

8321
CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG CTGTAGGTAT

8401
CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC

8481
CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA

8561
GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT

8641
ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG

8721
CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG

8801
GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC

8881
CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT

8961
CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA

9041
TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA

9121
ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG

9201
CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC

9281
GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA

9361
AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC

9441
ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG

9521
AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA

9601
GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC

9681
CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG

9761
CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT

9841
CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC

9921
CCGAAAAGTG CCACCTGACG TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC ACGAGGCCCT

10001
TTCGTCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA

10081
GCGGATGCCG GGAGCAGACA AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCTGGCTTA ACTATGCGGC

10161
ATCAGAGCAG ATTGTACTGA GAGTGCACCA TATCGACGCT CTCCCTTATG CGACTCCTGC ATTAGGAAGC AGCCCAGTAG

10241
TAGGTTGAGG CCGTTGAGCA CCGCCGCCGC AAGGAATGGT GCATGCAAGG AGATGGCGCC CAACAGTCCC CCGGCCACGG

10321
GGCCTGCCAC CATACCCACG CCGAAACAAG CGCTCATGAG CCCGAAGTGG CGAGCCCGAT CTTCCCCATC GGTGATGTCG

10401
GCGATATAGG CGCCAGCAAC CGCACCTGTG GCGCCGGTGA TGCCGGCCAC GATGCGTCCG GCGTAGAGGA TCTGGCTAGC

10481
GATGACCCTG CTGATTGGTT CGCTGACCAT TTCCGGGGTG CGGAACGGCG TTACCAGAAA CTCAGAAGGT TCGTCCAACC

10561
AAACCGACTC TGACGGCAGT TTACGAGAGA GATGATAGGG TCTGCTTCAG TAAGCCAGAT GCTACACAAT TAGGCTTGTA

10641
CATATTGTCG TTAGAACGCG GCTACAATTA ATACATAACC TTATGTATCA TACACATACG ATTTAGGTGA CACTATAGAA

10721
TACACCTGCA GGACGTCCCA ATGATCTTAA GTTAA

pCLD17 (SEQ ID NO: 29):

1
CCCTAGAAAG ATAATCATAT TGTGACGTAC GTTAAAGATA ATCATGCGTA AAATTGACGC ATGTGTTTTA TCGGTCTGTA

81
TATCGAGGTT TATTTATTAA TTTGAATAGA TATTAAGTTT TATTATATTT ACACTTACAT ACTAATAATA AATTCAACAA

161
ACAATTTATT TATGTTTATT TATTTATTAA AAAAAAACAA AAACTCAAAA TTTCTTCTAT AAAGTAACAA AACTTTTATC

241
GAATTTGCAG CCCGGGACTA GCTAGAGGGA CAGCCCCCCC CCAAAGCCCC CAGGGATGTA ATTACGTCCC TCCCCCGCTA

321
GGGGGCAGCA GCGAGCCGCC CGGGGCTCCG CTCCGGTCCG GCGCTCCCCC CGCATCCCCG AGCCGGCAGC GTGCGGGGAC

401
AGCCCGGGCA CGGGGAAGGT GGCACGGGAT CGCTTTCCTC TGAACGCTTC TCGCTGCTCT TTGAGCCTGC AGACACCTGG

481
GGGGATACGG GGAAAACTTA AGATCCGACC GGACGCGTAC TGAGAGCGCT ATTCTGAACT TTTCTTTTGT TCCCTTCCCT

561
TCTACCACAC CCTAATTGTA ATCCATTTTA ATTTCCTGGT CACAGTCCTG TCTCTCCTTC CATTGTACCT TGCCCTTTTC

641
TAAAGAGCGA CTGCAAAGTA TGTTTGCGTA GGTGAGGATC TAAAACTTTA TGAGGTACGA ACATCACAGA ATTACTTTGT

721
AATTTCAGTT TATTGTAGGC TTGGCTTTTT GGGGAGGGTT TACGTCTTAG ACCTCTTAGT GCTTCTTTGT TTCATGGTGT

801
TCTAACTTCG AAGCATCTCT GTAGCTTTAA TGGATTCCTT TTCTGAAAGC TTTGCTCTCT TTCTTCCCCC TCGGCTTTCT

881
CTTAGGCAAG AGGGCTAACT GTAAAGTAAG GCTTACTGCC TTGTGTTTCC AAATGTGTCC GAAGAGGAAG TGTCTTCTGT

961
GAATCCTGTT ATGCATGAAT AACAGGAAAT AGAAAGAAAT TCACTTTCAT TATTATAAAA GTAATATGTT CGTTTAAAAA

1041
ATTCTAATGA AGAGCTGGAG ATGCAACCCA GGGGTAGAGC ACACACTCAG CATGCAGGAG GCCCTGGGTC CAATCTTGGA

1121
ATCTCCTCTC AGTTAACCTG ATCTCTAGCT GATTAGTAGT GAGTGCAAGC CCACTTTCCT CTTCTGCCTC ATTGCTCAGT

1201
GATAACAGCT GTTAAACTTT GTCTTATTCT AAAACTACCT CTGTGCAAAT GCTAGCACAA TAATATATAT CATATGCACA

1281
TGATTTTTTT TTTATCTTGA AAAGTAAGTC AGTATAGCTA CAAAGTTCAC TTGGCATTGT CAACATTTCA CAGGCGTAAT

1361
ATTCCTCCTC TAGTACTGTC CTCTTCATTC TTTGTGACCA AGTTTGGAGA GAGTGCACAA ATGCCAGGGA GGTTTGTGGG

1441
AAGGTTTCTC ATGTTCTGGT AAGGCGAGTA AGAAAATAGT CTCATGCAGG TGAAATGAGT GCTATGCAGT ATATATTATA

1521
CCAGAGAACA GCAAATGACC AAATTCACAC TGAACTAGTT CAGTAAAATT GGCTTTGTCA AAGCTTTCCT TGCTTAAAAT

1601
GTAATTCCCT GTCATCCTAG TTCTGGTCTG GATTCTTTTC CTGGAGTCTT GACTTCCAGA TTCCCTGTGG ACTTTTGTTT

1681
GAGTTTCAAG CTTTTGAAAT ATAGAAACCT ATCTAACTTA ACAAACTTGG GAGAGAAAAG ACTCCAGAAC AACTGAAAAC

1761
AGACCAGGCT AAATGAATAG ACTTTATTCC TCTCTTCTTA CCTGCAGTTT TCAGATATGC AGAGTTGGAG CGGATCTTAG

1841
AGGTTGATTC ATTCATGCCT GAAGAAAACA CATTTTATAG ACCCTGTGCC CAAGTTCGTG GTGGACATCA CCCTTTATTT

1921
ACTAATTGCA CTACATAACA GGCATTTTAG AAGACTGCTC CAGTCAGAGA CCCCGCCTTA GAGGAATCTG TAAACCCTGA

2001
ACTCCTATCA CTCATGAGCA CTAGTTATGT TTGGAATGCC GTATTAAAAC AAAAGTTACA TTTCTAAACT TAAAATTTTC

2081
TAGCACAGAG ACAGTGGGAG TAGCTAACTT TGATAGACAT TTTTCTACTA AAAGTCTTTC TAAGTACATA ATCTTCTGTA

2161
AGTTGGAAAA CAGCAAAATA GAACGTCTCC TACGTAGTTA ATCTTTTTGC ATAATTTGCA CATGTAGGAG TTATTAGTAT

2241
ACGGGTAAGT TTTCACTTTT TCCCCCAACT GGAGTGTCTT GTGGCTGGGT TTGAAAAAGG GAACGGGAGG CCGCTGGAGG

2321
GGATTGGTAA ATGAGATAAA ACACCACTCA TTCAACTCAG TGACTCAGCA TTTAAATTTT CCATAAAAGG ATTAAAGGAA

2401
AATTAAACAA ATTCTTAAAG CCAAGACTCT GGAGAAACTT GTTGGTGTGC TTTAGTTTTC ACTGTTATGA CTCATGAATT

2481
TATGCATAAA TTAGTACATT TATAAAAACA TAGCCTTTTT AGAGTTTTCT GTTTGGCTAA AGTGCCATTG TTAGCATTTG

2561
GAATTACCTT TTTATGTCTT ATATTTTTTC CAAATAAAAA TAAATGTTTC TGCTGTCTTA CTACTGAAAC TACGTTGTGA

2641
GCACTTTAAA TTTCTCAAAG CAGTTTCGCC TGTTATACTT GGCGCTTAGT CATCGTCGTA CACAACAGGA CCTGATTAAG

2721
AAGGCTGTGC TGCCTCTAAG CCGGGCTAGA TTGTAGCCAC TAGCAACCAG GCTGCAATAA TTTCCCTTTG ATGACATCAT

2801
CCACTGTGGA AGAACCCAGT TGCTTCAGCC AGTCGAACTA TACAGTTCCA ACCTCATCAA ATATGGCATC TCCCTTGCCT

2881
GCTATAGCAG GGGGAGGAAA AAATGCCACC ATCTTTTTAA TCTAGCAAGC TTCTCTTTTC TTCATCTTTT TTTTTTTCTT

2961
TTAAAAAAAT TCTGATCATG GATGCTTCTT CCGATCCCTA TTTGCCTTAT GACGGGGGAG GAGACAATAT CCCCTTGAGG

3041
GAATTACATA AAAGAGGTAA GAGCATCCCC TTGCTCTGAA TCCTCTGTTG GTTGTTGTGC ATGCGGCTGG GCGGTTCTGG

3121
GGACAGGCTG TCTGTTGTCC TCTTGCTGCA ATGTGCTGCT TAGTTGCCCT GCCTTGTTGC TGTGGGAGAA TGCGACCTTC

3201
CCAGCAGGGC TGGCCCTCCC TGATTGTTTG CTCTGTGCAG ATTAGCCCTG CTTCAGATCA CATAGGGCTG CAGACTCCAT

3281
CTTCTGTGTG AAAATGCTTT CGGTTTGATT GCAGAAATAA GCTGCCTTTA CAGCCAGCTA AAGTCCTGGT GGTTGGTTGG

3361
CACCTGCAAA GTAGTATTTT TGTACCTCTG GAAACTTATA TTTTCTTTAC ACAGCAATAT CAAGTGCCGG TATGCCATTC

3441
TGTTTTGGCT GCTGCCAATT ACCATGTAGA CTTTGCACCA CAGAGTAATA GTAAAAGCTC CTAGCTGCAT TTTATAACAT

3521
TTAAAAATAG CAGGAAAGAA GAATTATTTT TGATTTAACA TGTTTTTGTC ATTTAACGTC TTAACTGATT GACATACTAT

3601
ATTGTCTGTC TCGTGGGTAT CTTGTACAAC TTGATAGGAT AAAGCAATTT AGTTTTTTTT TTTTTTTTTA AATACATCCA

3681
GAATGTAAGT CGTCAGTAGT TTTCGAACAG ATAAGTAATG GTGTTAATCT TTTGGCAGGC TTTGCCTTGG TCTCCTTAAA

3761
GCTAATTAGG TGTTACTTAA TTAAACTGCT CTTTTGCTCA TTTTCTTAAA TTATTTTTTT AAAAGATAGT TGGCATTTGC

3841
TGTTCTAGAA ATAAACTTCA AGAAACATTC TTTAGCCAGA TGACTTCATG TATGAGCCAT GTTAGTTTGA ATTATTTGCT

3921
TGGTGTTATA AACTTTATGG TTTAATACCA ACTTTTATTA TGTTTACAAG GTAAATAAGG AAAATTTCAA GTACATTTTG

4001
TATCCTGAGA ACAAATTTAA GTTCCATAGA ATTTAGGAAT TACAATGTAT TCAACAGATA CTTACTTGTC ATACTGTGCC

4081
TGCAAAACAA TAATTAGACT CTGAACAGGT GCAACAATTT TCTGTAGAAT TAGACAAGTC TTCTTTTGGC AGGTGTTACT

4161
AAGTAGGCCA TTTCCCAAGG AACAGGGAAT TTGCCAGGCT TTTGTGGTGG AGAGAATAGA ATGAATAAAT GCTGTGGGGA

4241
GTAAAGAGCT TGTCAGAAGA TGATTAGTTC TGTGGCACCA AAACCAAGAG ATCAGTTTTC CTGTGAGAAG TAAAGGAAGC

4321
ATTGTAGAAA AATAGATGTG TTGAAGTCTA CCGGTGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG

4401
ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC

4481
GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT

4561
GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT

4641
CTACGTATTA GTCATCGCTA TTACCATTGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC

4721
GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT

4801
CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCGTTTAGT

4881
GAACCGTCAG ATCTACCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGGTGAGT ACTCCCTCTC AAAAGCGGGC

4961
ATGACTTCTG CGCTAAGATT GTCAGTTTCC AAAAACGAGG AGGATTTGAT ATTCACCTGG CCCGCGGTGA TGCCTTTGAG

5041
GGTGGCCGCG TCCATCTGGT CAGAAAAGAC AATCTTTTTG TTGTCAAGCT TCCTTGATGA TGTCATACTT ATCCTGTCCC

5121
TTTTTTTTCC ACAGCTCGCG GTTGAGGACA AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC

5201
CGAACGGTAC TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCGGATCCGA ATTCATAGAT

5281
AACTGATCCA GTGCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG CCGGTGTGCG TTTGTCTATA TGTTATTTTC

5361
CACCATATTG CCGTCTTTTG GCAATGTGAG GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT

5441
CCCCTCTCGC CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG AAGACAAACA

5521
ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG GTGCCTCTGC GGCCAAAAGC CACGTGTATA

5601
AGATACACCT GCAAAGGCGG CACAACCCCA GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT

5681
CAAGCGTATT CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC TCGGTGCACA

5761
TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA CCACGGGGAC GTGGTTTTCC TTTGAAAAAC

5841
ACGATGGCCA CCTCAGCAAG TTCCCACTTG AACAAAAACA TCAAGCAAAT GTACTTGTGC CTGCCCCAGG GTGAGAAAGT

5921
CCAAGCCATG TATATCTGGG TTGATGGTAC TGGAGAAGGA CTGCGCTGCA AAACCCGCAC CCTGGACTGT GAGCCCAAGT

6001
GTGTAGAAGA GTTACCTGAG TGGAATTTTG ATGGCTCTAG TACCTTTCAG TCTGAGGGCT CCAACAGTGA CATGTATCTC

6081
AGCCCTGTTG CCATGTTTCG GGACCCCTTC CGCAGAGATC CCAACAAGCT GGTGTTCTGT GAAGTTTTCA AGTACAACCG

6161
GAAGCCTGCA GAGACCAATT TAAGGCACTC GTGTAAACGG ATAATGGACA TGGTGAGCAA CCAGCACCCC TGGTTTGGAA

6241
TGGAACAGGA GTATACTCTG ATGGGAACAG ATGGGCACCC TTTTGGTTGG CCTTCCAATG GCTTTCCTGG GCCCCAAGGT

6321
CCGTATTACT GTGGTGTGGG CGCAGACAAA GCCTATGGCA GGGATATCGT GGAGGCTCAC TACCGCGCCT GCTTGTATGC

6401
TGGGGTCAAG ATTACAGGAA CAAATGCTGA GGTCATGCCT GCCCAGTGGG AGTTCCAAAT AGGACCCTGT GAAGGAATCC

6481
GCATGGGAGA TCATCTCTGG GTGGCCCGTT TCATCTTGCA TCGAGTATGT GAAGACTTTG GGGTAATAGC AACCTTTGAC

6561
CCCAAGCCCA TTCCTGGGAA CTGGAATGGT GCAGGCTGCC ATACCAACTT TAGCACCAAG GCCATGCGGG AGGAGAATGG

6641
TCTGAAGCAC ATCGAGGAGG CCATCGAGAA ACTAAGCAAG CGGCACCGGT ACCACATTCG AGCCTACGAT CCCAAGGGGG

6721
GCCTGGACAA TGCCCGTCGT CTGACTGGGT TCCACGAAAC GTCCAACATC AACGACTTTT CTGCTGGTGT CGCCAATCGC

6801
AGTGCCAGCA TCCGCATTCC CCGGACTGTC GGCCAGGAGA AGAAAGGTTA CTTTGAAGAC CGCCGCCCCT CTGCCAATTG

6881
TGACCCCTTT GCAGTGACAG AAGCCATCGT CCGCACATGC CTTCTCAATG AGACTGGCGA CGAGCCCTTC CAATACAAAA

6961
ACTAATCTAG ATCCCCCTCG CTTTCTTGCT GTCCAATTTC TATTAAAGGT TCCTTTGTTC CCTAAGTCCA ACTACTAAAC

7041
TGGGGGATAT TATGAAGGGC CTTGAGCATC TGGATTCTGC CTAATAAAAA ACATTTATTT TCATTGCAAT GATGTATTTA

7121
AATTATTTCT GAATATTTTA CTAAAAAGGG AATGTGGGAG GTCAGTGCAT TTAAAACATA AAGAAATGAA GAGGGGGATC

7201
TTCGCGATAC TGCATCGATG AGGGACAGCC CCCCCCCAAA GCCCCCAGGG ATGTAATTAC GTCCCTCCCC CGCTAGGGGG

7281
CAGCAGCGAG CCGCCCGGGG CTCCGCTCCG GTCCGGCGCT CCCCCCGCAT CCCCGAGCCG GCAGCGTGCG GGGACAGCCC

7361
GGGCACGGGG AAGGTGGCAC GGGATCGCTT TCCTCTGAAC GCTTCTCGCT GCTCTTTGAG CCTGCAGACA CCTGGGGGGA

7441
TACGGGGAAA ATAGACACCG CGGTGGAGCT CCAGCTTTTG TTCCCTTTAG TGAGGGTTAA TTAGTTCTTA ATACGACTCA

7521
CTATAGGGCG AATTGGCTAC CGGGCCGCCC ATCGAGGGTA TCATAAGCTT ATATCTATAA CAAGAAAATA TATATATAAT

7601
AAGTTATCAC GTAAGTAGAA CATGAAATAA CAATATAATT ATCGTATGAG TTAAATCTTA AAAGTCACGT AAAAGATAAT

7681
CATGCGTCAT TTTGACTCAC GCGGTCGTTA TAGTTCAAAA TCAGTGACAC TTACCGCATT GACAAGCACG CCTCACGGGA

7761
GCTCCAAGCG GCGACTGAGA TGTCCTAAAT GCACAGCGAC GGATTCGCGC TATTTAGAAA GAGAGAGCAA TATTTCAAGA

7841
ATGCATGCGT CAATTTTACG CAGACTATCT TTCTAGGGTT AAATCGATAG ATGCGATCCT GCAGGTCTCC CTATAGTGAG

7921
TCGTATTAAT TTCGATAAGC CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT TGGGCGCTCT

8001
TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT

8081
ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA

8161
AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG

8241
CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC

8321
GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT

8401
CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC

8481
TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG

8561
GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG

8641
CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT

8721
TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA

8801
CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

8881
ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG

8961
GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA

9041
GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

9121
AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA

9201
GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC

9281
GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

9361
TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT

9441
AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG

9521
TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

9601
TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT

9681
GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA

9761
AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

9841
ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA

9921
GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT

10001
CGCGCGTTTC GGTGATGACG GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG

10081
CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCTGGC TTAACTATGC GGCATCAGAG

10161
CAGATTGTAC TGAGAGTGCA CCATATCGAC GCTCTCCCTT ATGCGACTCC TGCATTAGGA AGCAGCCCAG TAGTAGGTTG

10241
AGGCCGTTGA GCACCGCCGC CGCAAGGAAT GGTGCATGCA AGGAGATGGC GCCCAACAGT CCCCCGGCCA CGGGGCCTGC

10321
CACCATACCC ACGCCGAAAC AAGCGCTCAT GAGCCCGAAG TGGCGAGCCC GATCTTCCCC ATCGGTGATG TCGGCGATAT

10401
AGGCGCCAGC AACCGCACCT GTGGCGCCGG TGATGCCGGC CACGATGCGT CCGGCGTAGA GGATCTGGCT AGCGATGACC

10481
CTGCTGATTG GTTCGCTGAC CATTTCCGGG GTGCGGAACG GCGTTACCAG AAACTCAGAA GGTTCGTCCA ACCAAACCGA

10561
CTCTGACGGC AGTTTACGAG AGAGATGATA GGGTCTGCTT CAGTAAGCCA GATGCTACAC AATTAGGCTT GTACATATTG

10641
TCGTTAGAAC GCGGCTACAA TTAATACATA ACCTTATGTA TCATACACAT ACGATTTAGG TGACACTATA GAATACACCT

10721
GCAGGACGTC CCAATGATCT TAAGTTAA

5′ ITR (Virology 172(1), 156-169 (1989), GenBank: J04364.2, SEQ ID NO: 9):

ccctagaaagataatcatattgtgacgtacgttaaagataatcatgcgtaaaattgacgcatg

3′ ITR (Virology 172(1), 156-169 (1989), GenBank: J04364.2, SEQ ID NO: 10):

catgcgtcaattttacgcagactatctttctaggg

HS4 Insulator (Cell 74(3):505-14 (1993), Proc Natl Acad Sci USA. 94(2):575-80 (1997),

GenBank: U78775.2, SEQ ID NO: 11):

gagctcacggggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctagggggcagcagcgagccgcccggggct

ccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttc

ctctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaaagctttaggctgaaagagagatttagaatgacagaa

tcatagaacggcctgggttgcaaaggagcacagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggctgc

ccagagccacatccagcctggccttgaatgcctgcagggatggggcatccacagcctccttgggcaacctgttcagtgcgtcaccaccctctgg

gggaaaaactgcctcctcatatccaacccaaacctcccctgtctcagtgtaaagccattcccccttgtcctatcaagggggagtttgctgtgacatt

gttggtctggggtgacacatgtttgccaattcagtgcatcacggagaggcagatcttggggataaggaagtgcaggacagcatggacgtgggac

atgcaggtgttgagggctctgggacactctccaagtcacagcgttcagaacagccttaaggataagaagataggatagaaggacaaagagcaa

gttaaaacccagcatggagaggagcacaaaaaggccacagacactgctggtccctgtgtctgagcctgcatgtttgatggtgtctggatgcaag

cagaaggggtggaagagcttgcctggagagatacagctgggtcagtaggactgggacaggcagctggagaattgccatgtagatgttcataca

atcgtcaaatcatgaaggctggaaaagccctccaagatccccaagaccaaccccaacccacccaccgtgcccactggccatgtccctcagtgc

cacatccccacagttcttcatcacctccagggacggtgacccccccacctccgtgggcagctgtgccactgcagcaccgctctttggagaaggt

aaatcttgctaaatccagcccgaccctcccctggcacaacgtaaggccattatctctcatccaactccaggacggagtcagtgagaatatt

EASE (Cytotechnology 28(1-3):9-17 (1998), GenBank: AF193761.1, SEQ ID NO: 12):

gaattctgaacttttcttttgttcccttcccttctaccacaccctaattgtaatccattttaatttcctggtcacagtcctgtctctccttccattgtaccttgcc

cttttctaaagagcgactgcaaagtatgtttgcgtaggtgaggatctaaaactttatgaggtacgaacatcacagaattactttgtaatttcagtttattg

taggcttggctttttggggagggtttacgtcttagacctcttagtgcttctttgtttcatggtgttctaacttcgaagcatctctgtagctttaatggattcct

tttctgaaagctttgctctctttcttccccctcggctttctcttaggcaagagggctaactgtaaagtaaggcttactgccttgtgtttccaaatgtgtccg

aagaggaagtgtcttctgtgaatcctgttatgcatgaataacaggaaatagaaagaaattcactttcattattataaaagtaatatgttcgtttaaaaaat

tctaatgaagagctggagatgcaacccaggggtagagcacacactcagcatgcaggaggccctgggtccaatcttggaatctcctctcagttaa

cctgatctctagctgattagtagtgagtgcaagcccactttcctcttctgcctcattgctcagtgataacagctgttaaactttgtcttattctaaaactac

ctctgtgcaaatgctagcacaataatatatatcatatgcacatgattttttttttatcttgaaaagtaagtcagtatagctacaaagttcacttggcattgtc

aacatttcacaggcgtaatattcctcctctagtactgtcctcttcattctttgtgaccaagtttggagagagtgcacaaatgccagggaggtttgtggg

aaggtttctcatgttctggtaaggcgagtaagaaaatagtctcatgcaggtgaaatgagtgctatgcagtatatattataccagagaacagcaaatg

accaaattcacactgaactagttcagtaaaattggctttgtcaaagctttccttgcttaaaatgtaattccctgtcatcctagttctggtctggattcttttc

ctggagtcttgacttccagattccctgtggacttttgtttgagtttcaagcttttgaaatatagaaacctatctaacttaacaaacttgggagagaaaag

actccagaacaactgaaaacagaccaggctaaatgaatagactttattcctctcttcttacctgcagttttcagatatgcagagttggagcggatctta

gaggttgattcattcatgcctgaagaaaacacattttatagaccctgtgcccaagttcgtggtggacatcaccctttatttactaattgcactacataac

aggcattttagaagactgctccagtcagagaccccgccttagaggaatctgtaaaccctgaactcctatcactcatgagcactagttatgtttggaat

gccgtattaaaacaaaagttacatttctaaacttaaaattttctagcacagagacagtgggagtagctaactttgatagacatttttctactaaaagtctt

tctaagtacataatcttctgtaagttggaaaacagcaaaatagaacgtctcctacgtagttaatctttttgcataatttgcacatgtaggagttattagtat

acgggtaagttttcactttttcccccaactggagtgtcttgtggctgggtttgaaaaagggaacgggaggccgctggaggggattggtaaatgaga

taaaacaccactcattcaactcagtgactcagcatttaaattttccataaaaggattaaaggaaaattaaacaaattcttaaagccaagactctggag

aaacttgttggtgtgctttagttttcactgttatgactcatgaatttatgcataaattagtacatttataaaaacatagcctttttagagttttctgtttggctaa

agtgccattgttagcatttggaattacctttttatgtcttatattttttccaaataaaaataaatgtttctgctgtcttactactgaaactacgttgtgagcact

ttaaatttctcaaagcagtttcgcctgttatacttggcgcttagtcatcgtcgtacacaacaggacctgattaagaaggctgtgctgcctctaagccgg

gctagattgtagccactagcaaccaggctgcaataatttccctttgatgacatcatccactgtggaagaacccagttgcttcagccagtcgaactat

acagttccaacctcatcaaatatggcatctcccttgcctgctatagcagggggaggaaaaaatgccaccatctttttaatctagcaagcttctcttttct

tcatctttttttttttcttttaaaaaaattctgatcatggatgcttcttccgatccctatttgccttatgacgggggaggagacaatatccccttgagggaat

tacataaaagaggtaagagcatccccttgctctgaatcctctgttggttgttgtgcatgcggctgggcggttctggggacaggctgtctgttgtcctc

ttgctgcaatgtgctgcttagttgccctgccttgttgctgtgggagaatgcgaccttcccagcagggctggccctccctgattgtttgctctgtgcag

attagccctgcttcagatcacatagggctgcagactccatcttctgtgtgaaaatgctttcggtttgattgcagaaataagctgcctttacagccagct

aaagtcctggtggttggttggcacctgcaaagtagtatttttgtacctctggaaacttatattttctttacacagcaatatcaagtgccggtatgccattc

tgttttggctgctgccaattaccatgtagactttgcaccacagagtaatagtaaaagctcctagctgcattttataacatttaaaaatagcaggaaaga

agaattatttttgatttaacatgtttttgtcatttaacgtcttaactgattgacatactatattgtctgtctcgtgggtatcttgtacaacttgataggataaag

caatttagtttttttttttttttttaaatacatccagaatgtaagtcgtcagtagttttcgaacagataagtaatggtgttaatcttttggcaggctttgccttgg

tctccttaaagctaattaggtgttacttaattaaactgctcttttgctcattttcttaaattatttttttaaaagatagttggcatttgctgttctagaaataaact

tcaagaaacattctttagccagatgacttcatgtatgagccatgttagtttgaattatttgcttggtgttataaactttatggtttaataccaacttttattatg

tttacaaggtaaataaggaaaatttcaagtacattttgtatcctgagaacaaatttaagttccatagaatttaggaattacaatgtattcaacagatactt

acttgtcatactgtgcctgcaaaacaataattagactctgaacaggtgcaacaattttctgtagaattctgtgcttagtaaaaggttgctttttatattttg

agagaaatctatttaaagatcatggcccatattttgtgcatatttttttctgtataccatttccatatatgtgtgtgtgtgtacatatatgtatatatataaaat

gttagaacatttgaggaaatagctaaaagtacaaaagtaatgttttctaattttttactccccgaggttatttcttttttccttgttttcctttctctttgttccta

tcatcagtttctagtaatactcttattgaacagtgattattcaaatgtcacattatttattaatcagcatttaaatggtaaaaccagacagaccatactttct

ctgagtgatgacaacatccatttttagtaatgataaactagaagggtcaggcttgatagtctttgtcaggactaatttatagactgtaaaggccaaaag

aaataagaaatgtcaaaactcttgtgaaactagacatacagatattaccaagagagaaactagaaaaaaaaattctgtgacatggccttaatttgcc

aggcaccatcgtgaaggcctaaacccctcttagaagctcactcagatgccatcctgcttctctgatgagacttcctgtcaatacaaacatggtttag

gaagaatgagtgtttgcagtataaaccagttatttactagccttactttaagaatatactgtagtgtccttgagagagaaggtgtttgttttctgtaatttat

gacccttttgaaaccatagatcagcacaaaggaactggggatatggaaatgggaacataacttaaatccagaaaagtgaatcagattccctgtga

ggacaaaatgcaatatttagaaataggatctttaggctgggagggagaaaagaggaaaaaaatgaaagtataacatttttcataagtataagatttc

ataaaaaaatgaaatctataacatagagggtgttgataaagtaagcatggatatgtttagtaaagccgacagagctaagaattagctttgtgagtaat

tggacttaatcaaacttttcaaggtgggatacaaatgaataattgtagaataaatggataaaagaatatgaataaaatgaatagtgagtaaaaattaa

aaatgaagctttttacttaagtgcatattgtagtggctagaacaaatagattcaaaatagaaatcatttatatattcttgattagaagataaaatgttatttt

agaaatagccatctttggaagtaaatttgctatgttgaacaaccaggttttcataatttgtctcttattttttttcaggaagaaaaaaaaacttgacttattt

gtactgctaagttttattcaatgtgcttgcttgcttaaatttttaatgaagttttagtcatttggtggtcaaattccttttatctactaatcgcttttcgtggcttt

ggcttttaaaattgtatttactgcatttatttgtgtgtattaggagtcaggtggccatatgtgccatggcatgtgtatggaagcacttgtggacatgaatc

ttctctttccacatgtgtgggccactggaatcaaactagtgtcaggcttggcagcaatttttaatgcactgagccttctcaccaccccctggacttttgt

ggggcagaaggggacaagtttaatattttatttactccatgtagaaagcctttaaaaaatgtagaaagcctttaaactacctattgttttatttgaattatg

aagctcttgtgtttatataaattacagttaggtactgtggagactaatggtagctacaatagtaatattaatagctaaaacttagtagaatctgattgagt

taatttggccctttccatcataaggtactcttcccaagcatcacatgacctgtgcttaagtctggtgggggcttatggctttgatattgaaaacaaatcg

tcaaggatgttaatttcttgttactgctattacactgaattttctatggctctttaggagaggaagagacaagtcttcttttggcaggtgttactaagtagg

ccatttcccaaggaacagggaatttgccaggcttttgtggtggagagaatagaatgaataaatgctgtggggagtaaagagcttgtcagaagatg

attagttctgtggcaccaaaaccaagagatcagttttcctgtgagaagtaaaggaagcattgtagaaaaatagatgtgttgaagtct

Beta-globin polyA (Proc Natl Acad Sci USA 87(10):3924-8 (1990), GenBank: AH001475, SEQ

ID NO: 13):

tgccctggcccacaagtatcactaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatatt

atgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaatgatgtatttaaattatttctgaatattttactaaaaagggaatg

tgggaggtcagtgcatttaaaacataaagaaatgaagagctagttcaaaccttgggaaaatacactatatcttaaa

SV40 late polyA (Mol Cell Biol. 9(10):4248-58 (1989), GenBank: J02400.1, SEQ ID NO: 14):

cagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttattt

gtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaag

taaaacctctacaaatgtggta

SV40 promoter (Nature 273(5658):113-20 (1978), Proc. Natl. Acad. Sci. USA 81 (1):23-27 (1984),

GenBank: J02400.1, SEQ ID NO: 15):

tgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatcgct

gactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgca

aa

hCMV immediate-early enhancer/promoter (derived from GenBank X17403.1, SEQ ID NO: 16):

ggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccat

agtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagt

acgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgt

attagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccacccca

ttgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggc

gtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatc

TPL (SEQ ID NO: 17):

ctcttccgcatcgctgtctgcgagggccagctgttggggtgagtactccctctcaaaagcgggcatgacttctgcgctaagattgtcagtttccaaa

aacgaggaggatttgatattcacctggcccgcggtgatgcctttgagggtggccgcgtccatctggtcagaaaagacaatctttttgttgtcaagct

tccttgatgatgtcatacttatcctgtcccttttttttccacagctcgcggttgaggacaaactcttcgcggtctttccagtactcttggatcggaaaccc

gtcggcctccgaacggtactccgccaccgagggacctgagcgagtccgcatcgaccggatcggaaaacctc

hCMV promoter/intron in pCLD116 (derived from GenBank X17403.1, SEQ ID NO: 18):

gttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgcca

atagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctat

tgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcg

ctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaat

gggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtg

ggaggtctatataagcagagctcgtttagtgaaccgtcagatc

pUC replication origin On (SEQ ID NO: 19):

tttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggc

gtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgcttt

ctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcg

ccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgagg

tatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagtta

ccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaa

aaaaaggatctcaa

Ampicillin resistance gene (SEQ ID NO: 20):

ttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgg

gagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccgga

agggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaata

gtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcga

gttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggtt

atggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgc

ggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcgg

ggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagc

gtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcat

GS gene (SEQ ID NO: 21):

atggccacctcagcaagttcccacttgaacaaaaacatcaagcaaatgtacttgtgcctgccccagggtgagaaagtccaagccatgtatatctg

ggttgatggtactggagaaggactgcgctgcaaaacccgcaccctggactgtgagcccaagtgtgtagaagagttacctgagtggaattttgatg

gctctagtacctttcagtctgagggctccaacagtgacatgtatctcagccctgttgccatgtttcgggaccccttccgcagagatcccaacaagct

ggtgttctgtgaagttttcaagtacaaccggaagcctgcagagaccaatttaaggcactcgtgtaaacggataatggacatggtgagcaaccagc

acccctggtttggaatggaacaggagtatactctgatgggaacagatgggcaccatttggttggccttccaatggattcctgggccccaaggtc

cgtattactgtggtgtgggcgcagacaaagcctatggcagggatatcgtggaggctcactaccgcgcctgcttgtatgctggggtcaagattaca

ggaacaaatgctgaggtcatgcctgcccagtgggagttccaaataggaccctgtgaaggaatccgcatgggagatcatctctgggtggcccgttt

catcttgcatcgagtatgtgaagactttggggtaatagcaacctttgaccccaagcccattcctgggaactggaatggtgcaggctgccataccaa

ctttagcaccaaggccatgcgggaggagaatggtctgaagcacatcgaggaggccatcgagaaactaagcaagcggcaccggtaccacattc

gagcctacgatcccaaggggggcctggacaatgcccgtcgtctgactgggttccacgaaacgtccaacatcaacgacttttctgctggtgtcgcc

aatcgcagtgccagcatccgcattccccggactgtcggccaggagaagaaaggttactttgaagaccgccgcccctctgccaattgtgacccctt

tgcagtgacagaagccatcgtccgcacatgccttctcaatgagactggcgacgagcccttccaatacaaaaactaa

SV40 enhancer (SEQ ID NO: 22):

gctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaacca

ggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcc

catcccgcccctaactccgcccagttccgcccattctccgctccatcg

The coding sequence for the GOI was synthesized by Blue Heron Biotech, LLC, WA and inserted into pCLD116 (FIG. 2A), pCLD21 (FIG. 2B), pCLD22 (FIG. 2C), pCLD12 (FIG. 2D), or pCLD14 (FIG. 2E). In pCLD21-GOI, pCLD22-GOI, pCLD12-GOI, or pCLD14-GOI, the GOI coding sequence was driven by a hCMV immediate-early enhancer/promoter with adenovirus TPL. The eukaryotic selectable marker was connected with GOI through IRES and followed by a PolyA signal. Chicken β-globin HS4 insulator and piggyBac ITR were placed at the interface of bacteria backbone and mammalian expression cassettes. EASE was placed at 5′ of the hCMV immediate-early enhancer/promoter. Standard ampicillin resistant (AmpR) gene and pUC replication origin (Ori) were used for plasmid replication in E. coli.

For expression of multiple-chain recombinant proteins, such as monoclonal antibodies, a first expression vector encoding a first GOI (e.g., light chain) and a second expression vector encoding a second GOI (e.g., heavy chain) were co-transfected into mammalian host cells. The eukaryotic selection markers for the first expression vector and the second expression vector are different to ensure sufficient selection pressure for both of the polypeptides.

To compare with a commercially available expression vector, the coding sequence for the GOI was inserted into pEE14.4 (Lonza, U.K.), after digested with restriction enzymes, HindIII and EcoRI (New England Biolabs, MA).

Example 2: Host CHO Cell Lines

Various host CHO cell lines, including CHOK1SV™ (Lonza; Slough, U.K.), HD-BIOP1 (Horizon Discovery, U.K.), CHOZN® (Sigma-Aldrich, St. Louis, Mo.) and Merck proprietary GS knock-out CHO host cell lines CHO-1, CHO-2, and CHO-3, were used.

Example 3: Selection of Stably Transfected Cell Lines

Transposase mRNA was synthesized by using MEGAscript® T7 Kit (Thermo Fisher Scientific, MA). Plasmids were prepared by using QIAGEN kit (QIAGEN, Germany). Plasmid and Transposase mRNA were co-transfected into CHO host cells through electroporation. After the recovery period, transfected cells were selected in appropriate selection media, CD-CHO (Thermo Fisher Scientific, Waltham, Mass.). The stable cells were then evaluated using a shake flask fed-batch process when viability reached ˜90%. Cells were seeded at 0.5×10⁶viable cells/mL in chemically defined Dynamis™ medium (Thermo Fisher Scientific, Waltham, Mass.). Chemically defined feeding media, CellBoost 7a and Cell Boost 7b (GE Healthcare Life Sciences, Logan, Utah), were supplemented daily into culture together with D-glucose (Sigma-Aldrich, St. Louis, Mo.). Glucose and lactate levels were measured everyday using the RANDOX RX imola chemistry analyzer (Crumlin, UK). Cell density and viability were measured using a Beckman Coulter ViCELL cell counter (Beckman Coulter, Indianapolis, Ind.).

FIG. 3 demonstrates that all expression vectors illustrated in FIGS. 2A-2E reduced the selection time for stably transfected cells, compared to the control vector pEE14.4. The results showed that the selection stringency varies depending on the vector design. Without transposon technology, pCLD22 exhibited higher stringency than pCLD21 and the control vector (FIG. 3). However, with transposon technology, the stable recovery rates for pCLD116, pCLD12, and pCLD14 were significantly improved for up to two weeks, compared to the control vector, which suggests a higher genomic integration efficiency (FIG. 3). The reduction of selection time for stably transfected cells is independent of host cell lines (FIG. 4).

Example 4: Expression of a Monoclonal Antibody Using the Designed Expression Vectors

The light chain and the heavy chain of an exemplary monoclonal antibody were separately cloned into two expression vectors with the same design except the mammalian selection marker. Monoclonal antibody production levels, i.e., titers, were determined using Protein-A HPLC (Waters, MA).

FIG. 5 demonstrates that the expression of the monoclonal antibody highly depends on the vector design combining various regulatory elements. For the low expressing vector design, pCLD116, transposon technology increased the expression level by up to 10-fold (FIG. 5). But, with selected regulatory elements, including TPL, IRES, and EASE, pCLD21 was able to achieve similar expression level, compared to pCLD116 co-transfected with transposase mRNA (FIG. 5). In addition, by reducing the strength of IRES through deletion of nucleotides from its 3′ end, pCLD22 further increased the expression level by up to 5-fold (FIG. 5). On the other hand, transposon technology on top of regulatory elements, including TPL, IRES, and EASE, has marginal effect on further increasing the expression level, demonstrated by comparison between pCLD12 and pCLD21 and comparison between pCLD14 and pCLD22 (FIG. 5).

Example 5: Comparison Between the Effects of IRES-1 and IRES-3

Vectors pCLD12 and pCLD14 were selected to evaluate the effects of the nucleotide length of IRES on the level of antibody expression, quality of the antibody product, DNA and RNA copy numbers of the heavy chain (HC) and light chain (LC) in a stable clone.

HC and LC of an antibody were constructed into pCLD12 or pCLD14 with different length of IRES and IRES-linked selection marker glutamine synthetase (GS) or neomycin phosphotransferase (Neo). In FIG. 6, HC-GS represents the expression construct comprising HC, followed by IRES then GS selection marker; LC-Neo represents the expression construct comprising LC, followed by IRES then Neo selection marker; LC-GS represents the expression construct comprising LC, followed by IRES then GS selection marker; HC-Neo represents the expression construct comprising HC, followed by IRES then Neo selection marker.

A pair of HC expression construct and LC expression construct were co-transfected into CHO host cells, and the host cells were selected with a medium containing G418 and without glutamine until the cell viability reached 90%. The stably selected pools were evaluated for mAb production through fed-batch production. pCLD14 with IRES-3 demonstrated higher mAb titer (FIG. 6A) and higher specific productivity (protein produced per cell per day, FIG. 6B) than pCLD12 with IRES-1. Product quality was assessed by high molecular weight (HMW) aggregation level (FIG. 6C) and mannose 5 level (FIG. 6D). pCLD14 with IRES-3 demonstrated better or equivalent product qualities compared to pCLD12 with IRES-1.

Genomic DNA was extracted from the CHO cells using DNeasy Blood and Tissue Kit (QIAGEN, Germany). Total RNA from the CHO cells was extracted using RNeasy Plus Mini Kit (QIAGEN, Germany). cDNAs was prepared from the RNA samples by reverse transcription using SuperScript IV VILO Master Mix (Thermo Fisher Scientific, MA). Manufacturer-recommended protocols were followed for the extraction of DNA or RNA, and for RNA reverse transcription. QX200 Droplet Digital PCR (ddPCR) System (Bio-Rad, Hercules, Calif.) was performed to determine the copy number of the HC gene and the LC gene, and to quantify the transcript level of the HC mRNA and the LC mRNA. Fluorescently-labeled oligo nucleotide probes for the ddPCR reactions were designed using the Primer Express Software (Applied Biosystems, Thermo Fisher Scientific, MA) and synthesized by Invitrogen (Thermo Fisher Scientific, MA). FIGS. 7A and 7B show higher DNA copy numbers and higher transcript levels associated with pCLD14 than with pCLD12, which further demonstrates that vector pCLD14 with IRES-3 is a better choice than vector pCLD12 with IRES-1.

In summary, the above examples demonstrated that the vector configurations with innovative combination of regulatory elements result in shortened selection time for stable clones, increased DNA copy number and RNA transcript levels, increased protein expression, and improved product quality.

SEQUENCE LISTING

The present application is being filed with a computer readable form (CRF) copy of the Sequence Listing. The CRF entitled 24617WOPCT_SEQLIST.txt, which was created on Sep. 23, 2019 and is 141,567 bytes in size, is incorporated herein by reference in its entirety.

EXPRESSION VECTORS FOR EUKARYOTIC EXPRESSION SYSTEMS

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