Novel methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins

Abstract

The present invention relates to constructing libraries of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and collectively display at least a portion of the diversity of the family. In a preferred embodiment, the displayed polypeptides are human Fabs.

Description

BACKGROUND OF THE INVENTION

[0001] It is now common practice in the art to prepare libraries of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and collectively display at least a portion of the diversity of the family. In many common libraries, the displayed peptides, polypeptides or proteins are related to antibodies. Often, they are Fabs or single chain antibodies.

[0002] In general, the DNAs that encode members of the families to be displayed must be amplified before they are cloned and used to display the desired member on the surface of a genetic package. Such amplification typically makes use of forward and backward primers.

[0003] Such primers can be complementary to sequences native to the DNA to be amplified or complementary to oligonucleotides attached at the 5′ or 3′ ends of that DNA. Primers that are complementary to sequences native to the DNA to be amplified are disadvantaged in that they bias the members of the families to be displayed. Only those members that contain a sequence in the native DNA that is substantially complementary to the primer will be amplified. Those that do not will be absent from the family. For those members that are amplified, any diversity within the primer region will be suppressed.

[0004] For example, in European patent 368,684 B1, the primer that is used is at the 5′ end of the VH region of an antibody gene. It anneals to a sequence region in the native DNA that is said to be “sufficiently well conserved” within a single species. Such primer will bias the members amplified to those having this “conserved” region. Any diversity within this region is extinguished.

[0005] It is generally accepted that human antibody genes arise through a process that involves a combinatorial selection of V and J or V, D, and J followed by somatic mutations. Although most diversity occurs in the Complementary Determining Regions (CDRs), diversity also occurs in the more conserved Framework Regions (FRs) and at least some of this diversity confers or enhances specific binding to antigens (Ag). As a consequence, libraries should contain as much of the CDR and FR diversity as possible.

[0006] To clone the amplified DNAs for display on a genetic package of the peptides, polypeptides or proteins that they encode, the DNAs must be cleaved to produce appropriate ends for ligation to a vector. Such cleavage is generally effected using restriction endonuclease recognition sites carried on the primers. When the primers are at the 5′ end of DNA produced from reverse transcription of RNA, such restriction leaves deleterious 5′ untranslated regions in the amplified DNA. These regions interfere with expression of the cloned genes and thus the display of the peptides, polypeptides and proteins coded for by them.

SUMMARY OF THE INVENTION

[0007] It is an object of this invention to provide novel methods for constructing libraries of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and collectively display at least a portion of the diversity of the family. These methods are not biased toward DNAs that contain native sequences that are complementary to the primers used for amplification. They also enable any sequences that may be deleterious to expression to be removed from the amplified DNA before cloning and displaying.

[0008] It is another object of this invention to provide a method for cleaving single-stranded nucleic acid sequences at a desired location, the method comprising the steps of:

[0009] (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and

[0010] (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

[0011] the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

[0012] It is a further object of this invention to provide an alternative method for cleaving single-stranded nucleic acid sequences at a desired location, the method comprising the steps of:

[0013] (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type II-S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site; and

[0014] (ii) cleaving the nucleic acid solely at the cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

[0015] the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

[0016] It is another objective of the present invention to provide a method of capturing DNA molecules that comprise a member of a diverse family of DNAs and collectively comprise at least a portion of the diversity of the family. These DNA molecules in single-stranded form have been cleaved by one of the methods of this invention. This method involves ligating the individual single-stranded DNA members of the family to a partially duplex DNA complex. The method comprises the steps of:

[0017] (i) contacting a single-stranded nucleic acid sequence that has been cleaved with a restriction endonuclease with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region that remains after cleavage, the double-stranded region of the oligonucleotide including any sequences necessary to return the sequences that remain after cleavage into proper reading frame for expression and containing a restriction endonuclease recognition site 5′ of those sequences; and

[0018] (ii) cleaving the partially double-stranded oligonucleotide sequence solely at the restriction endonuclease recognition site contained within the double-stranded region of the partially double-stranded oligonucleotide.

[0019] It is another object of this invention to prepare libraries, that display a diverse family of peptides, polypeptides or proteins and collectively display at least part of the diversity of the family, using the methods and DNAs described above.

[0020] It is an object of this invention to screen those libraries to identify useful peptides, polypeptides and proteins and to use those substances in human therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 is a schematic of various methods that may be employed to amplify VH genes without using primers specific for VH sequences.

[0022] FIG. 2 is a schematic of various methods that may be employed to amplify VL genes without using VL sequences.

[0023] FIG. 3 depicts gel analysis of cleaved kappa DNA from Example 2.

[0024] FIG. 4 depicts gel analysis of cleaved kappa DNA from Example 2.

[0025] FIG. 5 depicts gel analysis of amplified kappa DNA from Example 2.

[0026] FIG. 6 depicts gel purified amplified kappa DNA from Example 2.

TERMS

[0027] In this application, the following terms and abbreviations are used:

Sense strand     The upper strand of ds DNA as
                 usually written. In the sense
                 strand, 5′-ATG-3′ codes for
                 Met.
Antisense strand The lower strand of ds DNA as
                 usually written. In the
                 antisense strand, 3′-TAC-5′
                 would correspond to a Met
                 codon in the sense strand.
Forward primer:  A “forward” primer is
                 complementary to a part of the
                 sense strand and primes for
                 synthesis of a new antisense-
                 strand molecule. “Forward
                 primer” and “lower-strand
                 primer” are equivalent.
Backward primer: A “backward” primer is
                 complementary to a part of the
                 antisense strand and primes
                 for synthesis of a new sense-
                 strand molecule. “Backward
                 primer” and “top-strand
                 primer” are equivalent.
Bases:           Bases are specified either by
                 their position in a vector or
                 gene as their position within
                 a gene by codon and base. For
                 example, “89.1” is the first
                 base of codon 89, 89.2 is the
                 second base of codon 89.
Sv               Streptavidin
Ap               Ampicillin
apR              A gene conferring ampicillin
                 resistance.
RE               Restriction endonuclease
URE              Universal restriction
                 endonuclease
Functionally     Two sequences are sufficiently
complementary    complementary so as to anneal
                 under the chosen conditions.
RERS             Restriction endonuclease
                 recognition site
AA               Amino acid
PCR              Polymerization chain reaction
GLGs             Germline genes
Ab               Antibody: an immunoglobin.
                 The term also covers any
                 protein having a binding
                 domain which is homologous to
                 an immunoglobin binding
                 domain. A few examples of
                 antibodies within this
                 definition are, inter alia,
                 immunoglobin isotypes and the
                 Fab, F(ab1)2, scfv, Fv, dAb and
                 Fd fragments.
Fab              Two chain molecule comprising
                 an Ab light chain and part of
                 a heavy-chain.
scFv             A single-chain Ab comprising
                 either VH::linker::VL or
                 VL::linker::VH
w.t.             Wild type
HC               Heavy chain
LC               Light chain
VK               A variable domain of a Kappa
                 light chain.
VH               A variable domain of a heavy
                 chain.
VL               A variable domain of a lambda
                 light chain.

[0028] In this application, all references referred to are specifically incorporated by reference.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The nucleic acid sequences that are useful in the methods of this invention, i.e., those that encode at least in part the individual peptides, polypeptides and proteins displayed on the genetic packages of this invention, may be naturally occurring, synthetic or a combination thereof. They may be mRNA, DNA or cDNA. In the preferred embodiment, the nucleic acids encode antibodies. Most preferably, they encode Fabs.

[0030] The nucleic acids useful in this invention may be naturally diverse, synthetic diversity may be introduced into those naturally diverse members, or the diversity may be entirely synthetic. For example, synthetic diversity can be introduced into one or more CDRs of antibody genes.

[0031] Synthetic diversity may be created, for example, through the use of TRIM technology (U.S. Pat. No. 5,869,644). TRIM technology allows control over exactly which amino-acid types are allowed at variegated positions and in what proportions. In TRIM technology, codons to be diversified are synthesized using mixtures of trinucleotides. This allows any set of amino acid types to be included in any proportion.

[0032] Another alternative that may be used to generate diversified DNA is mixed oligonucleotide synthesis. With TRIM technology, one could allow Ala and Trp. With mixed oligonucleotide synthesis, a mixture that included Ala and Trp would also necessarily include Ser and Gly. The amino-acid types allowed at the variegated positions are picked with reference to the structure of antibodies, or other peptides, polypeptides or proteins of the family, the observed diversity in germline genes, the observed somatic mutations frequently observed, and the desired areas and types of variegation.

[0033] In a preferred embodiment of this invention, the nucleic acid sequences for at least one CDR or other region of the peptides, polypeptides or proteins of the family are cDNAs produced by reverse transcription from mRNA. More preferably, the mRNAs are obtained from peripheral blood cells, bone marrow cells, spleen cells or lymph node cells (such as B-lymphocytes or plasma cells) that express members of naturally diverse sets of related genes. More preferable, the mRNAs encode a diverse family of antibodies. Most preferably, the mRNAs are obtained from patients suffering from at least one autoimmune disorder or cancer. Preferably, mRNAs containing a high diversity of autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphospholipid syndrome and vasculitis are used.

[0034] In a preferred embodiment of this invention, the cDNAs are produced from the mRNAs using reverse transcription. In this preferred embodiment, the mRNAs are separated from the cell and degraded using standard methods, such that only the full length (i.e., capped) mRNAs remain. The cap is then removed and reverse transcription used to produce the cDNAs.

[0035] The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., H J de Haard et al., Journal of Biological Chemistry, 274(26):18218-30 (1999). In the preferred embodiment of this invention where the mRNAs encode antibodies, primers that are complementary to the constant regions of antibody genes may be used. Those primers are useful because they do not generate bias toward subclasses of antibodies. In another embodiment, poly-dT primers may be used (and may be preferred for the heavy-chain genes). Alternatively, sequences complementary to the primer may be attached to the termini of the antisense strand.

[0036] In one preferred embodiment of this invention, the reverse transcriptase primer may be biotinylated, thus allowing the cDNA product to be immobilized on streptavidin (Sv) beads. Immobilization can also be effected using a primer labeled at the 5′ end with one of a) free amine group, b) thiol, c) carboxylic acid, or d) another group not found in DNA that can react to form a strong bond to a known partner on an insoluble medium. If, for example, a free amine (preferably primary amine) is provided at the 5′ end of a DNA primer, this amine can be reacted with carboxylic acid groups on a polymer bead using standard amide-forming chemistry. If such preferred immobilization is used during reverse transcription, the top strand RNA is degraded using well-known enzymes, such as a combination of RNAseH and RNAseA, either before or after immobilization.

[0037] The nucleic acid sequences useful in the methods of this invention are generally amplified before being used to display the peptides, polypeptides or proteins that they encode. Prior to amplification, the single-stranded DNAs may be cleaved using either of the methods described before. Alternatively, the single-stranded DNAs may be amplified and then cleaved using one of those methods.

[0038] Any of the well known methods for amplifying nucleic acid sequences may be used for such amplification. Methods that maximize, and do not bias, diversity are preferred. In a preferred embodiment of this invention where the nucleic acid sequences are derived from antibody genes, the present invention preferably utilizes primers in the constant regions of the heavy and light chain genes and primers to a synthetic sequence that are attached at the 5′ end of the sense strand Priming at such synthetic sequence avoids the use of sequences within the variable regions of the antibody genes. Those variable region priming sites generate bias against V genes that are either of rare subclasses or that have been mutated at the priming sites. This bias is partly due to suppression of diversity within the primer region and partly due to lack of priming when many mutations are present in the region complementary to the primer. The methods disclosed in this invention have the advantage of not biasing the population of amplified antibody genes for particular V gene types.

[0039] The synthetic sequences may be attached to the 5′ end of the DNA strand by various methods well known for ligating DNA sequences together. RT CapExtention is one preferred method.

[0040] In RT CapExtention (derived from Smart PCR(TM), a short overlap (5′- . . . GGG-3′ in the upper-strand primer (USP-GGG) complements 3′-CCC . . . 5′ in the lower strand) and reverse transcriptases are used so that the reverse complement of the upper-strand primer is attached to the lower strand.

[0041] In a preferred embodiment of this invention, the upper strand or lower strand primer may be also biotinylated or labeled at the 5′ end with one of a) free amino group, b) thiol, c) carboxylic acid and d) another group not found in DNA that can react to form a strong bond to a known partner as an insoluble medium. These can then be used to immobilize the labeled strand after amplification. The immobilized DNA can be either single or double-stranded.

[0042] FIG. 1 shows a schematic of the amplification of VH genes. FIG. 1, Panel A shows a primer specific to the poly-dT region of the 3′ UTR priming synthesis of the first, lower strand. Primers that bind in the constant region are also suitable. Panel B shows the lower strand extended at its 3′ end by three Cs that are not complementary to the mRNA. Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that hybridize to the 3′ terminal CCCs and extending the reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence. Panel D shows the result of PCR amplification using a 5′ biotinylated synthetic top-strand primer that replicates the 5′ end of the synthetic primer of panel C and a bottom-strand primer complementary to part of the constant domain. Panel E shows immobilized double-stranded (ds) cDNA obtained by using a 5′-biotinylated top-strand primer.

[0043] FIG. 2 shows a similar schematic for amplification of VL genes. FIG. 2, Panel A shows a primer specific to the constant region at or near the 3′ end priming synthesis of the first, lower strand. Primers that bind in the poly-dT region are also suitable. Panel B shows the lower strand extended at its 3′ end by three Cs that are not complementary to the mRNA. Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that hybridize to the 3′ terminal CCCs and extending the reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence. Panel D shows the result of PCR amplification using a 5′ biotinylated synthetic top-strand primer that replicates the 5′ end of the synthetic primer of panel C and a bottom-strand primer complementary to part of the constant domain. The bottom-strand primer also contains a useful restriction endonuclease site, such as AscI. Panel E shows immobilized ds cDNA obtained by using a 5′-biotinylated top-strand primer.

[0044] In FIGS. 1 and 2, each V gene consists of a 5′ untranslated region (UTR) and a secretion signal, followed by the variable region, followed by a constant region, followed by a 3′ untranslated region (which typically ends in poly-A). An initial primer for reverse transcription may be complementary to the constant region or to the poly A segment of the 3′-UTR. For human heavy-chain genes, a primer of 15 T is preferred. Reverse transcriptases attach several C residues to the 3′ end of the newly synthesized DNA. RT CapExtention exploits this feature. The reverse transcription reaction is first run with only a lower-strand primer. After about 1 hour, a primer ending in GGG (USP-GGG) and more RTase are added. This causes the lower-strand cDNA to be extended by the reverse complement of the USP-GGG up to the final GGG. Using one primer identical to part of the attached synthetic sequence and a second primer complementary to a region of known sequence at the 3′ end of the sense strand, all the V genes are amplified irrespective of their V gene subclass.

[0045] After amplification, the DNAs of this invention are rendered single-stranded. For example, the strands can be separated by using a biotinylated primer, capturing the biotinylated product on streptavidin beads, denaturing the DNA, and washing away the complementary strand. Depending on which end of the captured DNA is wanted, one will choose to immobilize either the upper (sense) strand or the lower (antisense) strand.

[0046] To prepare the single-stranded amplified DNAs for cloning into genetic packages so as to effect display of the peptides, polypeptides or proteins encoded, at least in part, by those DNAS, they must be manipulated to provide ends suitable for cloning and expression. In particular, any 5′ untranslated regions and mammalian signal sequences must be removed and replaced, in frame, by a suitable signal sequence that functions in the display host. Additionally, parts of the variable domains (in antibody genes) may be removed and replaced by synthetic segments containing synthetic diversity. The diversity of other gene families may likewise be expanded with synthetic diversity.

[0047] According to the methods of this invention, there are two ways to manipulate the single-stranded amplified DNAs for cloning. The first method comprises the steps of:

[0048] (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and

[0049] (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

[0050] the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

[0051] In this first method, short oligonucleotides are annealed to the single-stranded DNA so that restriction endonuclease recognition sites formed within the now locally double-stranded regions of the DNA can be cleaved. In particular, a recognition site that occurs at the same position in a substantial fraction of the single-stranded DNAs is identical.

[0052] For antibody genes, this can be done using a catalog of germline sequences. See, e.g., “http://www.mrc-cpe.cam.ac.uk/imt-doc/restricted/ok.html.” Updates can be obtained from this site under the heading “Amino acid and nucleotide sequence alignments.” For other families, similar comparisons exist and may be used to select appropriate regions for cleavage and to maintain diversity.

[0053] For example, Table 195 depicts the DNA sequences of the FR3 regions of the 51 known human VH germline genes. In this region, the genes contain restriction endonuclease recognition sites shown in Table 200. Restriction endonucleases that cleave a large fraction of germline genes at the same site are preferred over endonucleases that cut at a variety of sites. Furthermore, it is preferred that there be only one site for the restriction endonucleases within the region to which the short oligonucleotide binds on the single-stranded DNA, e.g., about 10 bases on either side of the restriction endonuclease recognition site.

[0054] An enzyme that cleaves downstream in FR3 is also more preferable because it captures fewer mutations in the framework. This may be advantageous is some cases. However, it is well known that framework mutations exist and confer and enhance antibody binding. The present invention, by choice of appropriate restriction site, allows all or part of FR3 diversity to be captured. Hence, the method also allows extensive diversity to be captured.

[0055] Finally, in the methods of this invention restriction endonucleases that are active between about 45° and about 75° C. are used. Preferably enzymes that are active above 50° C., and more preferably active about 55° C., are used. Such temperatures maintain the nucleic acid sequence to be cleaved in substantially single-stranded form.

[0056] Enzymes shown in Table 200 that cut many of the heavy chain FR3 germline genes at a single position include: MaeIII(24@4), Tsp45I(21@4), HphI(44@5), BsaJT(23@65), AluI(23@47), BlpI(21@48), DdeI(29@58), BglTI(10@61), MslI(44@72), BsiEI(23@74), EaeI(23@74), EagI(23@74), HaeIII(25@75), Bst4CI(51@86), HpyCH41II(51@86), HinfI(38@2), MlyI(18@2), PleI(18@2), MnlI(31@67), HpyCH4V(21@44), BsmAI(16@11), BpmI(19@12), XmnI(12@30), and SacI(11@51). (The notation used means, for example, that BsmAI cuts 16 of the FR3 germline genes with a restriction endonuclease recognition site beginning at base 11 of FR3.)

[0057] For cleavage of human heavy chains in FR3, the preferred restriction endonucleases are: Bst4CI (or TaaI or HpyCH41II), BlpI, HpyCH4V, and MslI. Because ACNGT (the restriction endonuclease recognition site for Bst4CI, TaaI, and HpyCH41II) is found at a consistent site in all the human FR3 germline genes, one of those enzymes is the most preferred for capture of heavy chain CDR3 diversity. BlpI and HpyCH4V are complementary. BlpI cuts most members of the VH1 and VH4 families while HpyCH4V cuts most members of the VH3, VH5, VH6, and VH7 families. Neither enzyme cuts VH2s, but this is a very small family, containing only three members. Thus, these enzymes may also be used in preferred embodiments of the methods of this invention.

[0058] The restriction endonucleases HpyCH4III, Bst4CI, and TaaI all recognize 5′-ACnGT-3′ and cut upper strand DNA after n and lower strand DNA before the base complementary to n. This is the most preferred restriction endonuclease recognition site for this method on human heavy chains because it is found in all germline genes. Furthermore, the restriction endonuclease recognition region (ACnGT) matches the second and third bases of a tyrosine codon (tay) and the following cysteine codon (tgy) as shown in Table 206. These codons are highly conserved, especially the cysteine in mature antibody genes.

[0059] Table 250 E shows the distinct oligonucleotides of length 22 (except the last one which is of length 20) bases. Table 255 C shows the analysis of 1617 actual heavy chain antibody genes. Of these, 1511 have the site and match one of the candidate oligonucleotides to within 4 mismatches. Eight oligonucleotides account for most of the matches and are given in Table 250 F.1. The 8 oligonucleotides are very similar so that it is likely that satisfactory cleavage will be achieved with only one oligonucleotide (such as H43.77.97.1-02#1) by adjusting temperature, pH, salinity, and the like. One or two oligonucleotides may likewise suffice whenever the germline gene sequences differ very little and especially if they differ very little close to the restriction endonuclease recognition region to be cleaved. Table 255 D shows a repeat analysis of 1617 actual heavy chain antibody genes using only the 8 chosen oligonucleotides. This shows that 1463 of the sequences match at least one of the oligonucleotides to within 4 mismatches and have the site as expected. Only 7 sequences have a second HpyCH4III restriction endonuclease recognition region in this region.

[0060] Another illustration of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of human heavy chains. Cleavage in FR1 allows capture of the entire CDR diversity of the heavy chain.

[0061] The germline genes for human heavy chain FR1 are shown in Table 217. Table 220 shows the restriction endonuclease recognition sites found in human germline genes FR1s. The preferred sites are BsgI(GTGCAG;39@4), BsoFI(GCngc;43@6,11@9,2@3,1@12), TseI (Gcwgc;43@6,11@9,2@3,1@12), MspA1I(CMGckg;46@7,2@1), PvuII(CAGctg;46@7,2@1), AluI(AGct;48@82@2), DdeI (Ctnag;22@52,9@48), HphI(tcacc;22@80), BssKI(Nccngg;35@39,2@40), BsaJI(Ccnngg;32@40,2@41), BstNI(CCwgg;33@40), ScrFI (CCngg;35@40,2@41), EcoO109I(RGgnccy;22@46, 11@43), Sau96I(Ggncc;23@47,11@44), AvaII(Ggwcc;23@47,4@44), PpuMI(RGgwccy;22@46,4@43), BsmFI(gtccc;20@48), HinfI(Gantc;34@16,21@56,21@77), TfiT(21@77), MlyI(GAGTC;34@16), MlyI(gactc;21@56), and AlwNI(CAGnnnctg;22@68). The more preferred sites are MspAI and PvuII. MspAI and PvuII have 46 sites at 7-12 and 2 at 1-6. To avoid cleavage at both sites, oligonucleotides are used that do not fully cover the site at 1-6. Thus, the DNA will not be cleaved at that site. We have shown that DNA that extends 3, 4, or 5 bases beyond a PvuII-site can be cleaved efficiently.

[0062] Another illustration of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of human kappa light chains. Table 300 shows the human kappa FR1 germline genes and Table 302 shows restriction endonuclease recognition sites that are found in a substantial number of human kappa FR1 germline genes at consistent locations. Of the restriction endonuclease recognition sites listed, BsmAI and PflFI are the most preferred enzymes. BsmAI sites are found at base 18 in 35 of 40 germline genes. PflFI sites are found in 35 of 40 germline genes at base 12.

[0063] Another example of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of the human lambda light chain. Table 400 shows the 31 known human lambda FR1 germline gene sequences. Table 405 shows restriction endonuclease recognition sites found in human lambda FR1 germline genes. HinfI and DdeI are the most preferred restriction endonucleases for cutting human lambda chains in FR1.

[0064] After the appropriate site or sites for cleavage are chosen, one or more short oligonucleotides are prepared so as to functionally complement, alone or in combination, the chosen recognition site. The oligonucleotides also include sequences that flank the recognition site in the majority of the amplified genes. This flanking region allows the sequence to anneal to the single-stranded DNA sufficiently to allow cleavage by the restriction endonuclease specific for the site chosen.

[0065] The actual length and sequence of the oligonucleotide depends on the recognition site and the conditions to be used for contacting and cleavage. The length must be sufficient so that the oligonucleotide is functionally complementary to the single-stranded DNA over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and solely at the desired location.

[0066] Typically, the oligonucleotides of this preferred method of the invention are about 17 to about 30 nucleotides in length. Below about 17 bases, annealing is too weak and above 30 bases there can be a loss of specificity. A preferred length is 18 to 24 bases.

[0067] Oligonucleotides of this length need not be identical complements of the germline genes. Rather, a few mismatches taken may be tolerated. Preferably, however, no more than 1-3 mismatches are allowed. Such mismatches do not adversely affect annealing of the oligonucleotide to the single-stranded DNA. Hence, the two DNAs are said to be functionally complementary.

[0068] The second method to manipulate the amplified single-stranded DNAs of this invention for cloning comprises the steps of:

[0069] (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type II-S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site; and

[0070] (ii) cleaving the nucleic acid solely at the cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

[0071] the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

[0072] This second method employs Universal Restriction Endonucleases (“URE”). UREs are partially double-stranded oligonucleotides. The single-stranded portion or overlap of the URE consists of a DNA adapter that is functionally complementary to the sequence to be cleaved in the single-stranded DNA. The double-stranded portion consists of a type II-S restriction endonuclease recognition site.

[0073] The URE method of this invention is specific and precise and can tolerate some (e.g., 1-3) mismatches in the complementary regions, i.e., it is functionally complementary to that region. Further, conditions under which the URE is used can be adjusted so that most of the genes that are amplified can be cut, reducing bias in the library produced from those genes.

[0074] The sequence of the single-stranded DNA adapter or overlap portion of the URE typically consists of about 14-22 bases. However, longer or shorter adapters may be used. The size depends on the ability of the adapter to associate with its functional complement in the single-stranded DNA and the temperature used for contacting the URE and the single-stranded DNA at the temperature used for cleaving the DNA with the type II-S enzyme. The adapter must be functionally complementary to the single-stranded DNA ver a large enough region to allow the two strands to associate such that the cleavage may occur at the chosen temperature and at the desired location. We prefer singe-stranded or overlap portions of 14-17 bases in length, and more preferably 18-20 bases in length.

[0075] The site chosen for cleavage using the URE is preferably one that is substantially conserved in the family of amplified DNAs. As compared to the first cleavage method of this invention, these sites do not need to be endonuclease recognition sites. However, like the first method, the sites chosen can be synthetic rather than existing in the native DNA. Such sites may be chosen by references to the sequences of known antibodies or other families of genes. For example, the sequences of many germline genes are reported at http://www.mrc-cpe.cam.ac.uk/imt-doc/restricted/ok.html. For example, one preferred site occurs near the end of FR3—codon 89 through the second base of codon 93. CDR3 begins at codon 95.

[0076] The sequences of 79 human heavy-chain genes are also available at http://www.ncbi.nlm.nih.gov/entre2/nucleotide.html. This site can be used to identify appropriate sequences for URE cleavage according to the methods of this invention. See, e.g., Table 8B.

[0077] Most preferably, one or more sequences are identified using these sites or other available sequence information. These sequences together are present in a substantial fraction of the amplified DNAs. For example, multiple sequences could be used to allow for known diversity in germline genes or for frequent somatic mutations. Synthetic degenerate sequences could also be used. Preferably, a sequence(s) that occurs in at least 65% of genes examined with no more than 2-3 mismatches is chosen

[0078] URE single-stranded adapters or overlaps are then made to be complementary to the chosen regions. Conditions for using the UREs are determined empirically. These conditions should allow cleavage of DNA that contains the functionally complementary sequences with no more than 2 or 3 mismatches but that do not allow cleavage of DNA lacking such sequences.

[0079] As described above, the double-stranded portion of the URE includes a Type II-S endonuclease recognition site. Any Type II-S enzyme that is active at a temperature necessary to maintain the single-stranded DNA substantially in that form and to allow the single-stranded DNA adapter portion of the URE to anneal long enough to the single-stranded DNA to permit cleavage at the desired site may be used.

[0080] The preferred Type II-S enzymes for use in the URE methods of this invention provide asymmetrical cleavage of the single-stranded DNA. Among these are the enzymes listed in Table 800. The most preferred Type II-S enzyme is FokI.

[0081] When the preferred Fok I containing URE is used, several conditions are preferably used to effect cleavage:

[0082] 1) Excess of the URE over target DNA should be present to activate the enzyme. URE present only in equimolar amounts to the target DNA would yield poor cleavage of ssDNA because the amount of active enzyme available would be limiting.

[0083] 2) An activator may be used to activate part of the FokI enzyme to dimerize without causing cleavage. Examples of appropriate activators are shown in Table 510.

[0084] 3) The cleavage reaction is performed at a temperature between 45°-75° C., preferably above 50° C. and most preferably above 55° C.

[0085] The UREs used in the prior art contained a 14-base single-stranded segment, a 10-base stem (containing a FokI site), followed by the palindrome of the 10-base stem. While such UREs may be used in the methods of this invention, the preferred UREs of this invention also include a segment of three to eight bases (a loop) between the FokI restriction endonuclease recognition site containing segments. In the preferred embodiment, the stem (containing the FokI site) and its palindrome are also longer than 10 bases. Preferably, they are 10-14 bases in length. Examples of these “lollipop” URE adapters are shown in Table 5.

[0086] One example of using a URE to cleave an single-stranded DNA involves the FR3 region of human heavy chain. Table 508 shows an analysis of 840 full-length mature human heavy chains with the URE recognition sequences shown. The vast majority (718/840=0.85) will be recognized with 2 or fewer mismatches using five UREs (VHS881-1.1, VHS881-1.2, VHS881-2.1, VHS881-4.1, and VHS881-9.1). Each has a 20-base adaptor sequence to complement the germline gene, a ten-base stem segment containing a FokI site, a five base loop, and the reverse complement of the first stem segment. Annealing those adapters, alone or in combination, to single-stranded antisense heavy chain DNA and treating with FokI in the presence of, e.g., the activator FOKIact, will lead to cleavage of the antisense strand at the position indicated.

[0087] Another example of using a URE(s) to cleave a single-stranded DNA involves the FR1 region of the human Kappa light chains. Table 512 shows an analysis of 182 full-length human kappa chains for matching by the four 19-base probe sequences shown. Ninety-six percent of the sequences match one of the probes with 2 or fewer mismatches. The URE adapters shown in Table 512 are for cleavage of the sense strand of kappa chains. Thus, the adaptor sequences are the reverse complement of the germline gene sequences. The URE consists of a ten-base stem, a five base loop, the reverse complement of the stem and the complementation sequence. The loop shown here is TTGTT, but other sequences could be used. Its function is to interrupt the palindrome of the stems so that formation of a lollypop monomer is favored over dimerization. Table 512 also shows where the sense strand is cleaved.

[0088] Another example of using a URE to cleave a single-stranded DNA involves the human lambda light chain. Table 515 shows analysis of 128 human lambda light chains for matching the four 19-base probes shown. With three or fewer mismatches, 88 of 128 (69%) of the chains match one of the probes. Table 515 also shows URE adapters corresponding to these probes. Annealing these adapters to upper-strand ssDNA of lambda chains and treatment with FokI in the presence of FOKIact at a temperature at or above 45° C. will lead to specific and precise cleavage of the chains.

[0089] The conditions under which the short oligonucleotide sequences of the first method and the UREs of the second method are contacted with the single-stranded DNAs may be empirically determined. The conditions must be such that the single-stranded DNA remains in substantially single-stranded form. More particularly, the conditions must be such that the single-stranded DNA does not form loops that may interfere with its association with the oligonucleotide sequence or the URE or that may themselves provide sites for cleavage by the chosen restriction endonuclease.

[0090] The effectiveness and specificity of short oligonucleotides (first method) and UREs (second method) can be adjusted by controlling the concentrations of the URE adapters/oligonucleotides and substrate DNA, the temperature, the pH, the concentration of metal ions, the ionic strength, the concentration of chaotropes (such as urea and formamide), the concentration of the restriction endonuclease(e.g., FokI), and the time of the digestion. These conditions can be optimized with synthetic oligonucleotides having: 1) target germline gene sequences, 2) mutated target gene sequences, or 3) somewhat related non-target sequences. The goal is to cleave most of the target sequences and minimal amounts of non-targets.

[0091] In the preferred embodiment of this invention, the single-stranded DNA is maintained in substantially that form using a temperature between 45° C. to 75° C. More preferably, a temperature between 50° C. and 60° C., most preferably between 55° C. and 60° C., is used. These temperatures are employed both when contacting the DNA with the oligonucleotide or URE and when cleaving the DNA using the methods of this invention.

[0092] The two cleavage methods of this invention have several advantages. The first method allows the individual members of the family of single-stranded DNAs to be cleaved solely at one substantially conserved endonuclease recognition site. The method also does not require an endonuclease recognition site to be built in to the reverse transcription or amplification primers. Any native or synthetic site in the family can be used.

[0093] The second method has both of these advantages. In addition, the URE method allows the single-stranded DNAs to be cleaved at positions where no endonuclease recognition site naturally occurs or has been synthetically constructed.

[0094] Most importantly, both cleavage methods permit the use of 5′ and 3′ primers so as to maximize diversity and then cleavage to remove unwanted or deleterious sequences before cloning and display.

[0095] After cleavage of the amplified DNAs using one of the methods of this invention, the DNA is prepared for cloning. This is done by using a partially duplexed synthetic DNA adapter, whose terminal sequence is based on the specific cleavage site at which the amplified DNA has been cleaved.

[0096] The synthetic DNA is designed such that when it is ligated to the cleaved single-stranded DNA, it allows that DNA to be expressed in the correct reading frame so as to display the desired peptide, polypeptide or protein on the surface of the genetic package. Preferably, the double-stranded portion of the adapter comprises the sequence of several codons that encode the amino acid sequence characteristic of the family of peptides, polypeptides or proteins up to the cleavage site. For human heavy chains, the amino acids of the 3-23 framework are preferably used to provide the sequences required for expression of the cleaved DNA.

[0097] Preferably, the double-stranded portion of the adapter is about 12 to 100 bases in length. More preferably, about 20 to 100 bases are used. The double-standard region of the adapter also preferably contains at least one endonuclease recognition site useful for cloning the DNA into a suitable display vector (or a recipient vector used to archive the diversity). This endonuclease restriction site may be native to the germline gene sequences used to extend the DNA sequence. It may be also constructed using degenerate sequences to the native germline gene sequences. Or, it may be wholly synthetic.

[0098] The single-stranded portion of the adapter is complementary to the region of the cleavage in the single-stranded DNA. The overlap can be from about 2 bases up to about 15 bases. The longer the overlap, the more efficient the ligation is likely to be. A preferred length for the overlap is 7 to 10. This allows some mismatches in the region so that diversity in this region may be captured.

[0099] The single-stranded region or overlap of the partially duplexed adapter is advantageous because it allows DNA cleaved at the chosen site, but not other fragments to be captured. Such fragments would contaminate the library with genes encoding sequences that will not fold into proper antibodies and are likely to be non-specifically sticky.

[0100] One illustration of the use of a partially duplexed adaptor in the methods of this invention involves ligating such adaptor to a human FR3 region that has been cleaved, as described above, at 5′-ACnGT-3′ using HpyCH4III, Bst4CI or TaaI.

[0101] Table 250 F.2 shows the bottom strand of the double-stranded portion of the adaptor for ligation to the cleaved bottom-strand DNA. Since the HpyCH4III-Site is so far to the right (as shown in Table 206), a sequence that includes the AflII-site as well as the XbaI site can be added. This bottom strand portion of the partially-duplexed adaptor, H43.XAExt, incorporates both XbaI and AflII-sites. The top strand of the double-stranded portion of the adaptor has neither site (due to planned mismatches in the segments opposite the XbaI and AflII-Sites of H43.XAExt), but will anneal very tightly to H43.XAExt. H43AExt contains only the AflII-site and is to be used with the top strands H43.ABr1 and H43.ABr2 (which have intentional alterations to destroy the AflII-site).

[0102] After ligation, the desired, captured DNA can be PCR amplified again, if desired, using in the preferred embodiment a primer to the downstream constant region of the antibody gene and a primer to part of the double-standard region of the adapter. The primers may also carry restriction endonuclease sites for use in cloning the amplified DNA.

[0103] After ligation, and perhaps amplification, of the partially double-stranded adapter to the single-stranded amplified DNA, the composite DNA is cleaved at chosen 5′ and 3′ endonuclease recognition sites.

[0104] The cleavage sites useful for cloning depend on the phage or phagemid into which the cassette will be inserted and the available sites in the antibody genes. Table 1 provides restriction endonuclease data for 75 human light chains. Table 2 shows corresponding data for 79 human heavy chains in each Table, the endonucleases are ordered by increasing frequency of cutting. In these Tables, Nch is the number of chains cut by the enzyme and Ns is the number of sites (some chains have more than one site).

[0105] From this analysis, SfiI, NotI, AflII, ApaLI, and AscI are very suitable. SfiI and NotI are preferably used in pCES1 to insert the heavy-chain display segment. ApaLI and AscI are preferably used in pCES1 to insert the light-chain display segment.

[0106] BstEII-sites occur in 97% of germ-line JH genes. In rearranged V genes, only 54/79 (68%) of heavy-chain genes contain a BstEII-Site and 7/61 of these contain two sites. Thus, 47/79 (59%) contain a single BstEII-Site. An alternative to using BstEII is to cleave via UREs at the end of JH and ligate to a synthetic oligonucleotide that encodes part of CH1.

[0107] One example of preparing a family of DNA sequences using the methods of this invention involves capturing human CDR 3 diversity. As described above, mRNAs from various autoimmune patients is reverse transcribed into lower strand cDNA. After the top strand RNA is degraded, the lower strand is immobilized and a short oligonucleotide used to cleave the cDNA upstream of CDR3. A partially duplexed synthetic DNA adapter is then annealed to the DNA and the DNA is amplified using a primer to the adapter and a primer to the constant region (after FR4). The DNA is then cleaved using BstEII (in FR4) and a restriction endonuclease appropriate to the partially double-stranded adapter (e.g., Xba I and AflII (in FR3)). The DNA is then ligated into a synthetic VH skeleton such as 3-23.

[0108] One example of preparing a single-stranded DNA that was cleaved using the URE method involves the human Kappa chain. The cleavage site in the sense strand of this chain is depicted in Table 512. The oligonucleotide kapextURE is annealed to the oligonucleotides (kaBR01UR, kaBR02UR, kaBR03UR, and kaBR04UR) to form a partially duplex DNA. This DNA is then ligated to the cleaved soluble kappa chains. The ligation product is then amplified using primers kapextUREPCR and CKForeAsc (which inserts a AscI site after the end of C kappa). This product is then cleaved with ApaLI and AscI and ligated to similarly cut recipient vector.

[0109] Another example involves the cleavage illustrated in Table 515. After cleavage, an extender (ON_LamEx133) and four bridge oligonucleotides (ON_LamB1-133, ON_LamB2-133, ON_LamB3-133, and ON_LamB4-133) are annealed to form a partially duplex DNA. That DNA is ligated to the cleaved lambda-chain sense strands. After ligation, the DNA is amplified with ON_Lam133PCR and a forward primer specific to the lambda constant domain, such as CL2ForeAsc or CL7ForeAsc (Table 130).

[0110] In human heavy chains, one can cleave almost all genes in FR4 (downstream, i.e. toward the 3′ end of the sense strand, of CDR3) at a BstEII-Site that occurs at a constant position in a very large fraction of human heavy-chain V genes. One then needs a site in FR3, if only CDR3 diversity is to be captured, in FR2, if CDR2 and CDR3 diversity is wanted, or in FR1, if all the CDR diversity is wanted. These sites are preferably inserted as part of the partially double-stranded adaptor.

[0111] The preferred process of this invention is to provide recipient vectors having sites that allow cloning of either light or heavy chains. Such vectors are well known and widely used in the art. A preferred phage display vector in accordance with this invention is phage MALIA3. This displays in gene III. The sequence of the phage MALIA3 is shown in Table 120A (annotated) and Table 120B (condensed).

[0112] The DNA encoding the selected regions of the light or heavy chains can be transferred to the vectors using endonucleases that cut either light or heavy chains only very rarely. For example, light chains may be captured with ApaLI and AscI. Heavy-chain genes are preferably cloned into a recipient vector having SfiI, NcoI, XbaI, AflII, BstEII, ApaI, and NotI sites. The light chains are preferably moved into the library as ApaLI-AscI fragments. The heavy chains are preferably moved into the library as SfiI-NotI fragments.

[0113] Most preferably, the display is had on the surface of a derivative of M13 phage. The most preferred vector contains all the genes of M13, an antibiotic resistance gene, and the display cassette. The preferred vector is provided with restriction sites that allow introduction and excision of members of the diverse family of genes, as cassettes. The preferred vector is stable against rearrangement under the growth conditions used to amplify phage.

[0114] In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed in a phagemid vector (e.g., pCES1) that displays the peptide, polypeptide or protein on the III protein. Such vectors may also be used to store the diversity for subsequent display using other vectors or phage.

[0115] In another embodiment, the mode of display may be through a short linker to three possible anchor domains. One anchor domain being the final portion of M13 III (“IIIstump”), a second anchor being the full length III mature protein, and the third being the M13 VIII mature protein.

[0116] The IIIstump fragment contains enough of M13 III to assemble into phage but not the domains involved in mediating infectivity. Because the w.t. III and VIII proteins are present, the phage is unlikely to delete the antibody genes and phage that do delete these segments receive only a very small growth advantage. For each of the anchor domains, the DNA encodes the w.t. AA sequence, but differs from the w.t. DNA sequence to a very high extent. This will greatly reduce the potential for homologous recombination between the display anchor and the w.t. gene that is also present.

[0117] Most preferably, the present invention uses a complete phage carrying an antibiotic-resistance gene (such as an ampicillin-resistance gene) and the display cassette. Because the w.t. iii and viii genes are present, the w.t. proteins are also present. The display cassette is transcribed from a regulatable promoter (e.g., PLacZ) Use of a regulatable promoter allows control of the ratio of the fusion display gene to the corresponding w.t. coat protein. This ratio determines the average number of copies of the display fusion per phage (or phagemid) particle.

[0118] Another aspect of the invention is a method of displaying peptides, polypeptides or proteins (and particularly Fabs) on filamentous phage. In the most preferred embodiment this method displays FABs and comprises:

[0119] a) obtaining a cassette capturing a diversity of segments of DNA encoding the elements:

[0120] Preq::RBS1::SS1::VL::CL::stop::RBS2::SS2::VH::CH1::linker::anchor::stop::,

[0121] where Preq is a regulatable promoter, RBS1 is a first ribosome binding site, SS1 is a signal sequence operable in the host strain, VL is a member of a diverse set of light-chain variable regions, CL is a light-chain constant region, stop is one or more stop codons, RBS2 is a second ribosome binding site, SS2 is a second signal sequence operable in the host strain, VH is a member of a diverse set of heavy-chain variable regions, CH1 is an antibody heavy-chain first constant domain, linker is a sequence of amino acids of one to about 50 residues, anchor is a protein that will assemble into the filamentous phage particle and stop is a second example of one or more stop codons; and

[0122] b) positioning that cassette within the phage genome to maximize the viability of the phage and to minimize the potential for deletion of the cassette or parts thereof.

[0123] The DNA encoding the anchor protein in the above preferred cassette should be designed to encode the same (or a closely related) amino acid sequence as is found in one of the coat proteins of the phage, but with a distinct DNA sequence. This is to prevent unwanted homologous recombination with the w.t. gene. In addition, the cassette should be placed in the intergenic region. The positioning and orientation of the display cassette can influence the behavior of the phage.

[0124] In one embodiment of the invention, a transcription terminator may be placed after the second stop of the display cassette above (e.g., Trp). This will reduce interaction between the display cassette and other genes in the phage antibody display vector (PADV).

[0125] In another embodiment of the methods of this invention, the phage or phagemid can display proteins other than Fab, by replacing the Fab portions indicated above, with other protein genes.

[0126] Various hosts can be used for growth of the display phage or phagemids of this invention. Such hosts are well known in the art. In the preferred embodiment, where Fabs are being displayed, the preferred host should grow at 30° C. and be RecA− (to reduce unwanted genetic recombination) and EndA− (to make recovery of RF DNA easier). It is also preferred that the host strain be easily transformed by electroporation.

[0127] XL1-Blue MRF′ satisfies most of these preferences, but does not grow well at 30° C. XL1-Blue MRF′ does grow slowly at 38° C. and thus is an acceptable host. TG-1 is also an acceptable host although it is RecA+ and EndA+. XL1-Blue MRF′ is more preferred for the intermediate host used to accumulate diversity prior to final construction of the library.

[0128] After display, the libraries of this invention may be screened using well known and conventionally used techniques. The selected peptides, polypeptides or proteins may then be used to treat disease. Generally, the peptides, polypeptides or proteins for use in therapy or in pharmaceutical compositions are produced by isolating the DNA encoding the desired peptide, polypeptide or protein from the member of the library selected. That DNA is then used in conventional methods to produce the peptide, polypeptides or protein it encodes in appropriate host cells, preferably mammalian host cells, e.g., CHO cells. After isolation, the peptide, polypeptide or protein is used alone or with pharmaceutically acceptable compositions in therapy to treat disease.

EXAMPLES

Example 1

Capturing Kappa Chains with BsmAI

[0129] A repertoire of human-kappa chain mRNAs was prepared by treating total or poly(A+) RNA isolated from a collection of patients having various autoimmune diseases with calf intestinal phosphatase to remove the 5′-phosphate from all molecules that have them, such as ribosomal RNA, fragmented mRNA, tRNA and genomic DNA. Full length mRNA (containing a protective 7-methyl cap structure) is unaffected. The RNA is then treated with tobacco acid pyrophosphatase to remove the cap structure from full length mRNAs leaving a 5′-monophosphate group.

[0130] Full length mRNA's were modified with an adaptor at the 5′ end and then reversed transcribed and amplified using the GeneRACE™ method and kit (Invitrogen). A 5′ biotinylated primer complementary to the adaptor and a 3′ primer complementary to a portion of the construct region were used.

[0131] Approximately 2 micrograms (ug) of human kappa-chain (Igkappa) gene RACE material with biotin attached to 5′-end of upper strand was immobilized on 200 microliters (μL) of Seradyn magnetic beads. The lower strand was removed by washing the DNA with 2 aliquots 200 μL of 0.1 M NaOH (pH 13) for 3 minutes for the first aliquot followed by 30 seconds for the second aliquot. The beads were neutralized with 200 μL of 10 mM Tris (pH 7.5) 100 mM NaCl. The short oligonucleotides shown in Table 525 were added in 40 fold molar excess in 100 μL of NEB buffer 2 (50 μM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol pH 7.9) to the dry beads. The mixture was incubated at 95° C. for 5 minutes then cooled down to 55° C. over 30 minutes. Excess oligonucleotide was washed away with 2 washes of NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol pH 7.9). Ten units of BsmAI (NEB) were added in NEB buffer 3 and incubated for 1 h at 55° C. The cleaved downstream DNA was collected and purified over a Qiagen PCR purification column (FIGS. 3 and 4).

[0132] A partially double-stranded adaptor was prepared using the oligonucleotide shown in Table 525. The adaptor was added to the single-stranded DNA in 100 fold molar excess along with 1000 units of T4 DNA ligase (NEB) and incubated overnight at 16° C. The excess oligonucleotide was removed with a Qiagen PCR purification column. The ligated material was amplified by PCR using the primers kapPCRt1 and kapfor shown in Table 525 for 10 cycles with the program shown in Table 530.

[0133] The soluble PCR product was run on a gel and showed a band of approximately 700 n, as expected (FIGS. 5 and 6). The DNA was cleaved with enzymes ApaLI and AscI, gel purified, and ligated to similarly cleaved vector pCES1. The presence of the correct size insert was checked by PCR in several clones as shown in FIG. 15.

[0134] Table 500 shows the DNA sequence of a kappa light chain captured by this procedure. Table 501 shows a second sequence captured by this procedure.

[0135] The closest bridge sequence was complementary to the sequence 5′-agccacc-3′, but the sequence captured reads 5′-Tgccacc-3′, showing that some mismatch in the overlapped region is tolerated.

Example 2

Construction of Synthetic CDR1 and CDR2 Diversity in V-3-23 VH Framework

[0136] A synthetic Complementary Determinant Region (CDR) 1 and 2 diversity was constructed in the 3-23 VH framework in a two step process: first, a vector containing the 3-23 VH framework was constructed, and then, a synthetic CDR 1 and 2 was assembled and cloned into this vector.

[0137] For construction of the V3-23 framework, 8 oligos and two PCR primers (long oligonucleotides: TOPFRLA, BOTFR1B, BOTFR2, BOTFR3, F06, BOTFR4, ON-vgC1, and ON-vgC2 and primers: SFPRMET and BOTPCRPRIM, shown in Table 600) that overlap were designed based on the Genebank sequence of V323 VH. The design incorporated at least one useful restriction site in each framework region, as shown in Table 600. In Table 600, the segments that were synthesized are shown as bold, the overlapping regions are underscored, and the PCR priming regions at each end are underscored. A mixture of these 8 oligos was combined at a final concentration of 2.5 uM in a 20 ul Polymerase Chain Reaction (PCR) reaction. The PCR mixture contained 200 uM dNTPs, 2.5 mM MgCl2, 0.02U Pfu TurboT DNA Polymerase, 1U Qiagen HotStart Taq DNA Polymerase, and 1×Qiagen PCR buffer. The PCR program consisted of 10 cycles of 94° C. for 30s, 55° C. for 30s, and 72° C. for 30s. The assembled V3-23 DNA sequence was then amplified, using 2.5 ul of a 10-fold dilution from the initial PCR in 100 ul PCR reaction. The PCR reaction contained 200 uM dNTPs, 2.5 mM MgCl2, 0.02U Pfu Turbo™ DNA Polymerase, 1U Qiagen HotStart Taq DNA Polymerase, 1×Qiagen PCR Buffer and 2 outside primers (SFPRMET and BOTPCRPRIM) at a concentration of 1 uM. The PCR program consisted of 23 cycles at 94° C. for 30 s, 55° C. for 30 s, and 72° C. for 60 s. The V3-23 VH DNA sequence was digested and cloned into pCES1 (phagemid vector) using the SfiI and BstEII restriction endonuclease sites (All restriction enzymes mentioned herein were supplied by New England BioLabs, Beverly, Mass. and used as per manufacturer's instructions).

[0138] Stuffer sequences (shown in Table 610 and Table 620) were introduced into pCES1 to replace CDR1/CDR2 sequences (900 bases between BspEI and XbaI RE sites) and CDR3 sequences (358 bases between AflII and BstEII), prior to cloning the CDR1/CDR2 diversity. The new vector is pCES5 and its sequence is given in Table 620. Having stuffers in place of the CDRs avoids the risk that a parental sequence would be over-represented in the library. The CDR1-2 stuffer contains restriction sites for BglII, Bsu36I, BclI, XcmI, MluI, PvuII, HpaI, and HincII, the underscored sites being unique within the vector pCES5. The stuffer that replaces CDR3 contains the unique restriction endonuclease site RsrII. The stuffer sequences are fragments from the penicillase gene of E. coli.

[0139] For the construction of the CDR1 and CDR2 diversity, 4 overlapping oligonucleotides (ON-vgCl, ON_Br12, ON_CD2Xba, and ON-vgC2, shown in Table 600 and Table 630) encoding CDR1/2, plus flanking regions, were designed. A mix of these 4 oligos was combined at a final concentration of 2.5 uM in a 40 ul PCR reaction. Two of the 4 oligos contained variegated sequences positioned at the CDR1 and the CDR2. The PCR mixture contained 200 uM dNTPs, 2.5U Pwo DNA Polymerase (Roche), and 1×Pwo PCR buffer with 2 mM MgSO4. The PCR program consisted of 10 cycles at 94° C. for 30 s, 60° C. for 30 s, and 72° C. for 60 s. This assembled CDR1/2 DNA sequence was amplified, using 2.5 ul of the mixture in 100 ul PCR reaction. The PCR reaction contained 200 uM dNTPs, 2.5U Pwo DNA Polymerase, 1×Pwo PCR Buffer with 2 mM MgSO4 and 2 outside primers at a concentration of 1 uM. The PCR program consisted of 10 cycles at 94° C. for 30 s, 60° C. for 30 s, and 72° C. for 60 s. These variegated sequences were digested and cloned into the V3-23 framework in place of the CDR1/2 stuffer.

[0140] We obtained approximately 7×107 independent transformants. Into this diversity, we can clone CDR3 diversity either from donor populations or from synthetic DNA.

[0141] It will be understood that the foregoing is only illustrative of the principles of this invention and that various modifications can be made by those skilled in the art without departing from the scope of and sprit of the invention.

TABLE 1
Cleavage of 75 human light chains.
				Planned location
Enzyme	Recognition*	Nch	Ns	of site
AfeI	AGCgct	0	0
AflII	Cttaag	0	0	HC FR3
AgeI	Accggt	0	0
AscI	GGcgcgcc	0	0	After LC
BglII	Agatct	0	0
BsiWI	Cgtacg	0	0
BSpDI	ATcgat	0	0
BssHII	Gcgcgc	0	0
BstBI	TTcgaa	0	0
DraIII	CACNNNgtg	0	0
EagI	Cggccg	0	0
FseI	GGCCGGcc	0	0
FspI	TGCgca	0	0
HpaI	GTTaac	0	0
MfeI	Caattg	0	0	HC FR1
MluI	Acgcgt	0	0
NcoI	Ccatgg	0	0	Heavy chain signal
NheI	Gctagc	0	0	HC/anchor linker
NotI	GCggccgc	0	0	In linker after HC
NruI	TCGcga	0	0
PacI	TTAATtaa	0	0
PmeI	GTTTaaac	0	0
PmlI	CACgtg	0	0
PvuI	CGATcg	0	0
SacII	CCGCgg	0	0
SalI	Gtcgac	0	0
SfiI	GGCCNNNNnggcc0	0	Heavy Chain signal
SgfI	GCGATcgc	0	0
SnaBI	TACgta	0	0
StuI	AGGcct	0	0
XbaI	Tctaga	0	0	HC FR3
AatII	GACGTc	1	1
AclI	AAcgtt	1	1
Aset	ATtaat	1	1
BsmI	GAATGCN	1	1
BspEI	Tccgga	1	1	HC FR1
BstXI	CCANNNNNntgg	1	1	HC FR2
DrdI	GACNNNNnngtc	1	1
HindIII	Aagctt	1	1
PciI	Acatgt	1	1
SapI	gaagagc	1	1
ScaI	AGTact	1	1
SexAI	Accwggt	1	1
SpeI	Actagt	1	1
TliI	Ctcgag	1	1
XhoI	Ctcgag	1	1
BcgI	cgannnnnntgc	2	2
BlpI	GCtnagc	2	2
BssSI	Ctcgtg	2	2
BstAPI	GCANNNNntgc	2	2
EspI	GCtnagc	2	2
KasI	Ggcgcc	2	2
PflMI	CCANNNNntgg	2	2
XmnI	GAANNnnttc	2	2
ApaLI	Gtgcac	3	3	LC signal seq
NaeI	GCCggc	3	3
NgoMI	Gccggc	3	3
PvuII	CAGctg	3	3
RsrII	CGgwccg	3	3
BsrBI	GAGcgg	4	4
BsrDI	GCAATGNNn	4	4
BstZl7I	GTAtac	4	4
EcoRI	Gaattc	4	4
SphI	GCATGc	4	4
SspI	AATatt	4	4
AccI	GTmkac	5	5
BclI	Tgatca	5	5
BsmBI	Nnnnnngagacg	5	5
BsrGI	Tgtaca	5	5
DraI	TTTaaa	6	6
NdeI	CAtatg	6	6	HC FR4
SwaI	ATTTaaat	6	6
BamHI Ggatcc	7	7
SacI	GAGCTc	7	7
BciVI	GTATCCNNNNNN	8	8
BsaBI	GATNNnnatc	8	8
NsiI	ATGCAt	8	8
Bsp120I	Gggccc	9	9	CH1
ApaI	GGGCCc	9	9	CH1
PsPOOMI	Gggccc	9	9
BspHI	Tcatga	9	11
ECoRV	GATatC	9	9
AhdI	GACNNNnngtc	11	11
BbsI	GAAGAC	11	14
PsiI	TTAtaa	12	12
BsaI	GGTCTCNnnnn	13	15
XmaI	Cccggg	13	14
AvaI	Cycgrg	14	16
BglI	GCCNNNNnggc	14	17
AlwNI	CAGNNNctg	16	16
BspMI	ACCTGC	17	19
XcmI	CCANNNNNnnnntgg	17	26
BstEII	Ggtnacc	19	22	HC FR4
Sse8387I	CCTGCAgg	20	20
AvrII	Cctagg	22	22
HincII	GTYrac	22	22
BsgI	GTGCAG	27	29
MscI	TGGcca	30	34
BseRI	NNnnnnnnnnctcctc	32	35
Bsu36I	CCtnagg	35	37
PstI	CTGCAg	35	40
EciI	nnnnnnnnntccgcc	38	40
PpuMI	RGgwccy	41	50
StyI	Ccwwgg	44	73
Eco0109I	RGgnccy	46	70
Acc65I	Ggtacc	50	51
KpnI	GGTACc	50	51
BpmI	ctccag	53	82
AvaII	Ggwcc	71	124
*cleavage occurs in the top strand after the last upper-case base. For REs that cut palindromic sequences, the lower strand is cut at the symmetrical site.

[0142]

TABLE 2
Cleavage of 79 human heavy chains
					Planned location
	Enzyme	Recognition	Nch	Ns	of site
	AfeI	AGCgct	0	0
	AflII	Cttaag	0	0	HC FR3
	AscI	GGcgcgcc	0	0	After LC
	BsiWI	Cgtacg	0	0
	BspDI	ATcgat	0	0
	BssHII	Gcgcgc	0	0
	FseI	GGCCGGcc	0	0
	HpaI	GTTaac	0	0
	NheI	Gctagc	0	0	HC Linker
	NotI	GCggccgc	0	0	In linker, HC/
					anchor
	NruI	TCCcga	0	0
	NsiI	ATGCAt	0	0
	PacI	TTAATtaa	0	0
	PciI	Acatgt	0	0
	PmeI	GTTTaaac	0	0
	PvuI	CGATcg	0	0
	RsrII	CGgwccg	0	0
	SapI	gaagagc	0	0
	SfiI	GGCCNNNNnggcc	0	0	HC signal seq
	SgfI	GCGATcgc	0	0
	SwaI	ATTTaaat	0	0
	AclI	AAcgtt	1	1
	AgeI	Accggt	1	1
	AseI	ATtaat	1	1
	AvrII	Cctagg	1	1
	BsmI	GAATGCN	1	1
	BsrBI	GAGcgg	1	1
	BsrDI	GCAATGNNn	1	1
	DraI	TTTaaa	1	1
	FspI	TGCgca	1	1
	HindIII	Aagctt	1	1
	MfeI	Caattg	1	1	HC FR1
	NaeI	GCCggc	1	1
	NgoMI	Gccggc	1	1
	SpeI	Actagt	1	1
	Acc65I	Ggtacc	2	2
	BstBI	TTcgaa	2	2
	KpnI	GGTACc	2	2
	MluI	Acgcgt	2	2
	NcoI	Ccatgg	2	2	In HC signal seq
	NdeI	CAtatg	2	2	HC FR4
	PmlI	CACgtg	2	2
	XcmI	CCANNNNNnnnntgg	2	2
	BcgI	cgannnnnntgc	3	3
	BclI	Tgatca	3	3
	BglI	GCCNNNNnggc	3	3
	BsaBI	GATNNnnatc	3	3
	BsrGI	Tgtaca	3	3
	SnaBI	TACgta	3	3
	Sse8387I	CCTGCAgg	3	3
	ApaLI	Gtgcac	4	4	LC Signal/FR1
	BspHI	Tcatga	4	4
	BssSI	Ctcgtg	4	4
	PsiI	TTAtaa	4	5
	SphI	GCATGc	4	4
	AhdI	GACNNNnngtc	5	5
	BspEI	Tccgga	5	5	HC FR1
	MscI	TGGcca	5	5
	SacI	GAGCTc	5	5
	ScaI	AGTact	5	5
	SexAI	Accwggt	5	6
	SspI	AATatt	5	5
	TliI	Ctcgag	5	5
	XhoI	Ctcgag	5	5
	BbsI	GAAGAC	7	8
	BstAPI	GCANNNNntgc	7	8
	BstZl7I	GTAtac	7	7
	EcoRV	GATatc	7	7
	EcoRI	Gaattc	8	8
	BlpI	GCtnagc	9	9
	Bsu36I	CCtnagg	9	9
	DraIII	CACNNNgtg	9	9
	EspI	Gctriagc	9	9
	StuI	AGGcct	9	13
	XbaI	Tctaga	9	9	HC FR3
	Bsp120I	Gggccc	10	11	CH1
	ApaI	GGGCCC	10	11	CH1
	PspOOMI	Gggccc	10	11
	BciVI	GTATCCNNNNNN	11	11
	SalI	Gtcgac	11	12
	DrdI	GACNNNNnngtc	12	12
	KasI	Ggcgcc	12	12
	XmaI	Cccggg	12	14
	BglII	Agatct	14	14
	HincII	GTYrac	16	18
	BamHI	Ggatcc	17	17
	PflMI	CCANNNNntgg	17	18
	BsmBI	Nnnnnngagacg	18	21
	BstXI	CCANNNNNntgg	18	19	HC FR2
	XmnI	GAANNnnttc	18	18
	SacII	CCGCgg	19	19
	PstI	CTGCAg	20	24
	PvuII	CAGctg	20	22
	AvaI	Cycgrg	21	24
	EagI	Cggccg	21	22
	AatII	GACGTc	22	22
	BspMI	ACCTGC	27	33
	AccI	GTmkac	30	43
	StyI	Ccwwgg	36	49
	AlwNI	CAGNNNctg	38	44
	BsaI	GGTCTCNnnnn	38	44
	PpuMI	RGgwccy	43	46
	BsgI	GTGCAG 44	54
	BseRI	NNnnnnnnnnctcctc	48	60
	EciI	nnnnnnnnntccgcc	52	57
	BstEII	Ggtnacc	54	61	HC Fr4, 47/79 have
					one
	EcoOl09I	RGgnccy	54	86
	BpmI	ctccag	60	121
	AvaII	Ggwcc	71	140

[0143]

TABLE 5
Use of FokI as “Universal Restriction Enzyme”
1
2
3
4

[0144]

TABLE 8
Matches to URE FR3 adapters in 79 human HC.
A. List of Heavy-chains genes sampled
AF008566
AF035043
AF103026
af103033
AF103061
AF103072
af103078
AF103099
AF103102
AF103103
AF103174
AF103186
af103187
AF103195
af103277
af103286
AF103309
af103343
AF103367
AF103368
AF103369
AF103370
af103371
AF103372
AF158381
E05213
E05886
E05887
HSA235661
HSA235664
HSA235660
HSA235659
HSA235678
HSA235677
HSA235676
HSA235675
HSA235674
HSA235673
HSA240559
HSCB201
HSIGGVHC
HSU44791
HSU44793
HSU82771
HSU82949
HSU82950
HSU82952
HSU82961
HSU86522
HSU86523
HSU92452
HSU94412
HSU94415
HSU94416
HSU94417
HSU94418
HSU96389
HSU96391
HSU96392
HSU96395
HSZ93849
HSZ93850
HSZ93851
HSZ93853
HSZ93855
HSZ93857
HSZ93860
HSZ93863
MCOMFRAA
MCOMFRVA
S82745
S32764
S83240
SABVH369
SADEIGVH
SAH2IGVH
SDA3IGVH
SIGVHTTD
SUK4IGVH

[0145]

TABLE 8 B
Testing all distinct GLGs from bases 89.1 to 93.2 of the heavy
variable domain
Id	Nb	0	1	2	3	4			SEQ ID NO:
1	38	15	11	10	0	2	Seq1	gtgtattactgtqc	25
2	19	7	6	4	2	0	Seq2	gtAtattactgtgc	26
3	1	0	0	1	0	0	Seq3	gtgtattactgtAA	27
4	7	1	5	1	0	0	Seq4	gtgtattactgtAc	28
5	0	0	0	0	0	0	Seq5	Ttgtattactgtgc	29
6	0	0	0	0	0	0	Seq6	TtgtatCactgtgc	30
7	3	1	0	1	1	0	Seq7	ACAtattactgtgc	31
8	2	0	2	0	0	0	Seq8	ACgtattactgtgc	32
9	9	2	2	4	1	0	Seq9	ATgtattactcTtQc	33
Group	26	26	21	4	2
Cumulative	26	52	73	77	79

[0146]

TABLE 8C
Most important URE recognition seqs in FR3 Heavy
1	VHSzy1	GTGtattactgtgc	(ON_SHC103)	(SEQ ID NO:25)
2	VHSzy2	GTAtattactgtgc	(ON_SHC323)	(SEQ ID NO:26)
3	VHSzy4	GTGtattactgtac	(ON_SHC349)	(SEQ ID NO:28)
4	VHSzy9	ATctattactgtgc	(ON_SHC5a)	(SEQ ID NO:33)

[0147]

TABLE 8D
testing 79 human HC V genes with four probes
Number of sequences . . .    79
Number of bases . . . 29143
	Number of mismatches
Id	Best	0	1	2	3	4	5
1	39	15	11	10	1	2	0	Seq1	gtgtattactgtgc	(SEQ ID NO:25)
2	22	7	6	5	3	0	1	Seq2	gtAtattactgtgc	(SEQ ID NO:26)
3	7	1	5	1	0	0	0	Seq4	gtgtattactgtAc	(SEQ ID NO:28)
4	11	2	4	4	1	0	0	Seq9	ATgtattactgtgc	(SEQ ID NO:33)
Group	25	26	20	5	2
Cumulative	25	51	71	76	78

[0148] One sequence has five mismatches with sequences 2, 4, and 9; it is scored as best for 2.

[0149] Id is the number of the adapter.

[0150] Best is the number of sequence for which the identified adapter was the best available.

[0151] The rest of the table shows how well the sequences match the adapters. For example, there are 11 sequences that match VHSzy1(Id=1) with 2 mismatches and are worse for all other adapters. In this sample, 90% come within 2 bases of one of the four adapters.

TABLE 130
PCR primers for amplification of human Ab genes
(HuIgMFOR)      5′-tgg aag agg cac gtt ctt ttc ttt-3′
!(HuIgMFOREtop)5′-aaa gaa aag aac gtg cct ctt cca-3′=reverse complement
(HuCkFOR)       5′-aca ctc tcc cct gtt gaa gct ctt-3′
(HuCL2FOR)      5′-tga aca ttc tgt agg ggc cac tg-3′
(HuCL7FOR)      5′-aga gca ttc tgc agg ggc cac tg-3′
! Kappa
(CKForeAsc)  5′-acc gcc tcc acc ggg cgc gcc tta tta aca ctc tcc cct gtt-
gaa gct ctt-3′
(CL2ForeAsc)    5′-acc gcc tcc acc ggg cgc gcc tta tta tga aca ttc tgt-
agg ggc cac
(CL7ForeAsc)    5′-acc gcc tcc acc ggg cgc gcc tta tta aga gca ttc tgc-
agg ggc cac tg-3′

[0152]

TABLE 195
Human GLG FR3 sequences
! VH1
! 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
agg gtc acc atg acc agg gac acg tcc atc agc aca gcc tac atg
81  82  82a 82b 82c 83  84  85  86  87  88  89  90  91  92
gag ctg agc agg ctg aga tct gac gac acg gcc gtg tat tac tgt
93  94  95
gcg aga ga ! 1-02# 1
aga gtc acc att acc agg gac aca tcc gcg agc aca gcc tac atg
gag ctg agc agc ctg aga tct gaa gac acg gct gtg tat tac tgt
gcg aga ga ! 1-03# 2
aga gtc acc atg acc agg aac acc tcc ata agc aca gcc tac atg
gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt
gcg aga gg ! 1-08# 3
aga gtc acc atg acc aca gac aca tcc acg agc aca gcc tac atg
gag ctg agg agc ctg aga tct gac gac acg gcc gtg tat tac tgt
gcg aga ga ! 1-18# 4
aga gtc acc atg acc gag gac aca tct aca gac aca gcc tac atg
gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt
gca aca ga ! 1-24# 5
aga gtc acc att acc agg gac agg tct atg agc aca gcc tac atg
gag ctg agc agc ctg aga tct gag gac aca gcc atg tat tac tgt
gca aga ta ! 1-45# 6
aga gtc acc atg acc agg gac acg tcc acg agc aca gtc tac atg
gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt
gcg aga ga ! 1-46# 7
aga gtc acc att acc agg gac atg tcc aca agc aca gcc tac atg
gag ctg agc agc ctg aga tcc gag gac acg gcc gtg tat tac tgt
gcg gca ga ! 1-58# 8
aga gtc acg att acc gcg gac gaa tcc acg agc aca gcc tac atg
gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt
gcg aga ga ! 1-69#9
aga gtc acg att acc gcg gac aaa tcc acg agc aca gcc tac atg
gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt
gcg aga ga ! 1-3# 10
aga gtc acc ata acc gcg gac acg tct aca gac aca gcc tac atg
gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt
gca aca ga ! 1-f# 11
VH2
agg ctc acc atc acc aag gac acc tcc aaa aac cag gtg gtc ctt
aca atg acc aac atg gac cct gtg gac aca gcc aca tat tac tgt
gca cac aga c! 2-05# 12
agg ctc acc atc tcc aag gac acc tcc aaa agc cag gtg gtc ctt
acc atg acc aac atg gac cct gtg gac aca gcc aca tat tac tgt
gca cgg ata c! 2-05# 12
agg ctc acc atc tcc aag gac acc tcc aaa agc cag gtg gtc ctt
acc atg acc aac atg gac cct gtg gac aca gcc aca tat tac tgt
gca cgg ata c! 2-26# 13
agg ctc acc atc tcc aag gac acc tcc aaa aac cag gtg gtc ctt
aca atg acc aac atg gac cct gtg gac aca gcc acg tat tac tgt
gca cgg ata c! 2-70# 14
VH3
cga ttc aac atc tcc aga gac aac gcc aag aac tca ctg tat ctg
caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt
gcg aga ga ! 3-07# 15
cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg
caa atg aac agt ctg aga gct gag gac acg gcc ttg tat tac tgt
gca aaa gat 1! 3-09#16
cga ttc acc atc tcc agg gac aac gcc aag aac tca ctg tat ctg
caa atg aac agc ctg aga gcc gag gac acg gcc gtg tat tac tgt
gcg aga ga ! 3-11# 17
cga ttc acc atc tcc aga gaa aat gcc aag aac tcc ttg tat ctt
caa atg aac agc ctg aga gcc ggg gac acg gct gtg tat tac tgt
gca aga ga ! 3-13# 18
aga ttc acc atc tca aga gat gat tca aaa aac acg ctg tat ctg
caa atg aac agc ctg aaa acc gag gac aca ggg gtg tat tac tgt
acc aca ga ! 3-15# 19
cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg
caa atg aac agt ctg aga gcc gag gac acg gcc ttg tat cac tgt
gcg aga ga ! 3-20# 20
cga ttc acc atc tcc aga gac aac gcc aag aac tca ctg tat ctg
caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt
gcg aga ga ! 3-21# 21
cgg ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg
caa atg aac agc ctg aga gcc gag gac acg gcc gta tat tac tgt
gcg aaa ga ! 3-23# 22
cga ttc aac atc tcc aga gac aat tcc aag aac acg ctg tat ctg
caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt
gcg aaa ga ! 3-30# 23
cga ttc aac atc tcc aga gac aat tcc aag aac acg ctg tat ctg
caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt
gcg aga ga ! 3303# 24
cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg
caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt
gcg aga ga ! 3305# 25
cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg
caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt
gcg aga ga ! 3-33# 26
cga ttc acc atc tcc aga gac aac agc aaa aac tcc ctg tat ctg
caa atg aac agt ctg aga act gag gac acc gcc ttg tat tac tgt
gca aaa gat a! 3-43#27
cga ttc acc atc tcc aga gac aat gcc aag aac tca ctg tat ctg
caa atg aac agc ctg aga gac gag gac acg gct gtg tat tac tgt
gcg aga ga ! 3-48# 28
aga ttc acc atc tca aga gat ggt tcc aaa agc atc gcc tat ctg
caa atg aac agc ctg aaa acc gag gac aca gcc gtg tat tac tgt
act aga ga ! 3-49# 29
cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctt
caa atg aac agc ctg aga gcc gag gac acg gcc gtg tat tac tgt
gcg aga ga ! 3-53# 30
aga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctt
caa atg ggc agc ctg aga gct gag gac atg gct gtg tat tac tgt
gcg aga ga ! 3-64# 31
aga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctt
caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt
gcg aga ga ! 3-66# 32
aga ttc acc atc tca aga gat gat tca aag aac tca ctg tat ctg
caa atg aac agc ctg aaa acc gag gac acg gcc gtg tat tac tgt
gct aga ga ! 3-72# 33
agg ttc acc atc tcc aga gat gat tca aag aac acg gcg tat ctg
caa atg aac agc ctg aaa acc gag gac acg gcc ctg tat tac tgt
act aga ca · 3-73# 34
cga ttc acc atc tcc aga gac aac gcc aag aac acg ctg tat ctg
caa atg aac agt ctg aga gcc gag gac acg gct gtg tat tac tgt
gca aga ga ! 3-74# 35
aga ttc acc atc tcc aga gac aat tcc aag aac acg ctg cat ctt
caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt
aag aaa ga ! 3-d#36
VH4
cga gtc acc ata tca gta gac aag tcc aag aac cag ttc tcc ctg
aag ctg agc tct gtg acc gcc gcg gac acg gcc gtg tac tac tgt
gcg aga ga ! 4-04# 37
cga gtc acc atg tca gta gac acg tcc aag aac cag ttc tcc ctg
aag ctg agc tct gtg acc gcc gtg gac acg gcc gtg tat tac tgt
gcg aga aa ! 4-28 # 38
cga gtt acc ata tca gta gac acg tct aag aac cag ttc tcc ctg
aag ctg agc tct gtg act gcc gcg gac acg gcc gtg tat tac tgt
gcg aga ga ! 4301# 39
cga gtc acc ata tca gta gac agg tcc aag aac cag ttc tcc ctg
aag ctg agc tct gtg acc gcc gcg gac acg gcc gtg tat tac tgt
gcc aga ga ! 4302# 40
cga gtt acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg
aag ctg agc tct gtg act gcc gca gac acg gcc gtg tat tac tgt
gcc aga ga ! 4304# 41
cga gtt acc ata tca gta gac acg tct aag aac cag ttc tcc ctg
aag ctg agc tct gtg act gcc gcg gac acg gcc gtg tat tac tgt
gcg aga ga ! 4-31# 42
cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg
aag ctg agc tct gtg acc gcc gcg gac acg gct gtg tat tac tgt
gcg aga ga !4-34# 43
cga gtc acc ata tcc gta gac acg tcc aag aac cag ttc tcc ctg
aag ctg agc tct gtg acc gcc gca gac acg gt gtg tat tac tgt
gcg aga ca ! 4-39#10 44
cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg
aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat tac tgt
gcg aga ga ! 4-59# 45
cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg
aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat tac tgt
gcg aga ga ! 4-61# 46
cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg
aag ctg agc tct gtg acc gcc gca gac acg gcc gtg tat tac tgt
gcg aga ga ! 4-b# 47
! VH5
cag gtc acc atc tca gcc gac aag tcc atc agc acc gcc tac ctg
cag tgg agc agc ctg aag gcc tcg gac acc gcc atg tat tac tgt
gcg aga ca ! 5-51# 48
cac gtc acc atc tca gct gac aag tcc atc agc act gcc tac ctg
cag tgg agc agc ctg aag gcc tcg gac acc gcc atg tat tac tgt
gcg aga  ! 5-a# 49
! VH6
cga ata acc atc aac cca gac aca tcc aag aac cag ttc tcc ctg
cag ctg aac tct gtg act ccc gag gac acg gct gtg tat tac tgt
gca aga ga ! 6-1# 50
! VH7
cgg ttt gtc ttc tcc ttg gac acc tct gtc agc acg gca tat ctg
cag atc tgc agc cta aag gct gag gac act gcc gtg tat tac tgt
gcg aga ga ! 71.1# 51

[0153]

TABLE 250
REdaptors, Extenders, and Bridges used for Cleavage and Capture of
Human Heavy Chains in FR3.
A: BpYCH4V Probes of actual human BC genes
!HpyCH4V in FR3 of human HC, bases35-56; only those with TGca site
TGca; 10,
RE recognition:tgca	of length 4 is expected at 10
1	6-1	agttctccctgcagctgaactc
2	3-11, 3-07, 3-21, 3-72, 3-48	cactgtatctgcaaatgaacag
3	3-09, 3-43, 3-20	ccctgtatctgcaaatgaacag
4	5-51	ccgcctacctgcagtggagcag
5	3-15, 3-30, 3-30.5, 2-30.3, 3-74, 3-23, 3-33	cgctgtatctgcaaatgaacag
6	7-4.1	cggcatatctgcagatctgcag
7	3-73	cggcgtatctgcaaatgaacag
8	5-a	ctgcctacctgcagtggagcag
9	3-49	tcgcctatctgcaaatgaacag
B: HpyCH4V REdaptors, Extenders, and Bridges
B.1 REdaptors
! Cutting HC lower strand:
! TmKeller for 100 mM NaCl, zero formamide
! Edapters for cleavage		Tm W	Tm K
(ON_HCFR36-1)	5′-agttctcccTGCAgctgaactc-3′	68.0	64.5
(ON_HCFR36-1A)	5′-ttctcccTGCAgctgaactc-3′	62.0	62.5
(ON_HCFR36-1B)	5′-ttctcccTGCAgctgaac-3′	56.0	59.9
(ON_HCFR33-15)	5′-cgctgtatcTGCAaatgaacag-3′	64.0	60.8
(ON_HCFR33-15A)	5′-ctgtatcTGCAaatgaacag-3′	56.0	56.3
(ON_HCFR33-15B)	5′-ctgtatcTGCAaatgaac-3′	50.0	53.1
(ON_HCER33-11)	5′-cactgtatcTcCAaatgaacag-3′	62.0	58.9
(ON_HCFR35-51)	5′-ccgcctaccTGCAgtggagcag-3′	74.0	70.1
!
B.2 Segment of synthetic 3-23 gene into which captured CDR3 is to be cloned
!                    XbaI...
!D323*  cgCttcacTaag tcT aga gac aaC tcT aag aaT acT ctC taC
!       scab........ designed gene 3-23 gene.................
!
!    HpyCH4V
!     .. ..           AflII...
!    Ttg caG atg aac agc TtA agG . . .
!    ............................ . . .
!
B.3 Extender and Bridges
!Extender (bottom strand):
!
(ON_HCHpyEx01)  5′-cAAgTAgAgAgTATTcTTAgAgTTgTcTcTAgAcTTAgTgAAgcg-3′
! ON — HCHpyEx01 is the reverse complement of
! 5′-cgCttcacTaag tcT aga gac aaC tcT aag aaT acT ctC taC Ttg-3′
!
! Bridges (top strand, 9-base overlap):
!
(ON_HCHpyBr016-1)   5′-cgCttcacTaag tCT aga gac aaC tcT aag-
aaT acT ctC taC Ttg CAgctgaac-3′ {3′-term C is blocked}
!
! 3-15 et al. + 3-11
(ON_HCHpyBr023-15) 5′-cgCttcacTaag tcT aga gac aaC tcT aag-
aaT acT ctC taC Ttg CAaatgaac-3′ {3′-term C is blocked}
!
! 5-51
(ON_HCHpyBr045-51)  5′-cgCttcacTaag tcT aga gac aaC tcT aag-
aaT acT ctC taC Ttg CAgtggagc-3′ {3′-term C is blocked}
!
! PCR primer (top strand)
!
(ON_HCHpyPCR)        5′-cgCttcacTaag tcT aga gac-3′
!
C: BlpI Probes from human HC GLGs
1	1-58,1-03,1-08,1-69,1-24,1-45,1-46,1-f, 1−e	acatggaGCTGAGCagcctgag
2	1-02	acatggaGCTGAGCaagctgag
3	1-18	acatggagctgaggagcctgag
4	5-51,5-a	acctgcagtggagcagcctgaa
5	3-15,3-73,3-49,3-72	atctgcaaatgaacagcctgaa
6	3303,3-33,3-07,3-11,3-30,3-21,3-23,3305,3-48	atctgcaaatgaacagcctgag
7	3-20,3-74,3-09, 3-43	atctgcaaatgaacagtctgag
8	74.1	atctgcagatctgcagcctaaa
9	3-66, 3-13, 3-53, 3-d	atcttcaaatgaacagcctgag
10	3-64	atcttcaaatgggcagcctgag
11	4301,4-28,4302,4-04,4304,4-31,4-34,4-39,4-59,4-61,4-b	ccctgaaGCTGAGCtctgtgac
12	6-1	ccctgcagctgaactctgtgac
13	2-70,2-05	tccttacaatgaccaacatgga
14	2-26	tccttaccatgaccaacatgga
D: BlpI REdaptors, Extenders, and Bridges
D.1 REdaptors
	Tm W	Tm K
(BlpF3HC1-58)	5′-ac atg gaG CTG AGC agc ctg ag-3′	70	66.4
(BlpF3HC6-1)	5′-cc ctg aag ctg agc tct gtg ac-3′	70	66.4
! BlpF3HC6-1	matches 4-30.1, not 6-1.
D.2 Segment of synthetic 3 -23 gene into which captured CDR3 is to be cloned
!                                                                 BlpI
!                    XbaI...                                       . ... ...
!D323*  cgCttcacTaag TCT AGA gac aaC tcT aag aaT acT ctC taC Ttg caG atg aac
!
!                    AflII...
!                 agC TTA AGG
D.3 Extender and Bridges
! Bridges
(BlpF3Brl) 5′-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG-
tac Ttg caG Ctg a|GC agc ctg-3′
(BlpF3Br2) 5′-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG-
taC Ttg caG Ctg a|gc tct gtg-3′
| lower strand is cut here
!
! Extender
(BlpF3Ext) 5′-
TcAgcTgcAAgTAcAAAgTATTTTTAcTgTTATcTcTAgAcTgAgTgAAgcg-3′
! BlpF3Ext is the reverse complement of:
! 5′-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG taC Ttg caG Ctg a-3′
!
(BlpF3PCR) 5′-cgCttcacTcag tcT aga gaT aaC-3′
E: HpyCH4III  Distinct GLG sequences surrounding site, bases 77-98
1	102#1,118#4,146#7,169#9,1e#10,311#17,353#30,404#37,4301	ccgtgtattactgtgcgagaga
2	103#2, 307#15, 321#21, 3303#24,333#26,348#28, 364#31,366#32	ctgtgtattactgtgcgagaga
3	108#3	ccgtgtattactgtgcgagagg
4	124#5,1f#11	ccgtgtattactgtgcaacaga
5	145#6	ccatgtattactgtgcaagata
6	158#8	ccgtgtattactgtgcggcaga
7	205#12	ccacatattactgtgcacacag
8	226#13	ccacatattactgtgcacggat
9	270#14	ccacgtattactgtgcacggat
10	309#16,343#27	ccttgtattactgtgcaaaaga
11	313#18,374#35,61#50	ctgtgtattactgtgcaagaga
12	315#19	ccgtgtattactgtaccacaga
13	320#20	ccttgtatcactgtgcgagaga
14	323#22	ccgtatattactgtgcgaaaga
15	330#23,3305#25	ctgtgtattactgtgcgaaaga
16	349#29	ccgtgtattactgtactagaga
17	372#33	ccgtgtattactgtgctagaga
18	373#34	ccgtgtattactgtactagaca
19	3d#36	ctgtgtattactgtaagaaaga
20	428#38	ccgtgtattactgtgcgagaaa
21	4302#40,4304#41	ccgtgtattactgtgccagaga
22	439#44	ctgtgtattactgtgcgagaca
23	551#48	ccatgtattactgtgcgagaca
24	5a#49	ccatgtattactgtgcgaga
F: HpyCH4III REdaptors, Extenders, and Bridges
F.1 REdaptors
! ONs for cleavage of HC(lower) in FR3(bases 77-97)
! For cleavage with HpyCH4III, Bst4CI, or TaaI
! cleavage is in lower chain before base 88.
!	77 788 888 888 889 999 999 9
!	78 901 234 567 890 123 456 7	Tm W	Tm K
(H43.77.97.1-02#1)	5′-cc gtg tat tAC TGT gcg aga g-3′	64	62.6
(H43.77.97.1-03#2)	5′-ct gtg tat tAC TGT gcg aga g-3′	62	60.6
(H43.77.97.108#3)	5′-cc gtg tat tAC TGT gcg aga g-3′	64	62.6
(H43.77.97.323#22)	5′-cc gta tat tac tgt gcg aaa g-3′	60	58.7
(H43.77.97.330#23)	5′-ct gtg tat tac tgt gcg aaa g-3′	60	58.7
(H43.77.97.439#44)	5′-ct gtg tat tac tgt gcg aga c-3′	62	60.6
(H43.77.97.551#48)	5′-cc atg tat tac tgt gcg aga c-3′	62	60.6
(H43.77.97.5a#49)	5′-cc atg tat tAC TGT gcg aga  3′	58	58.3
F.2 Extender and Bridges
! XbaI and AflII Sites in bridges are bunged
(H43.XABr1) 5′-ggtgtagtga-
|TCT|AGt|gac|aac|tct|aag|aat|act|ctc|tac|55g|cag|atg|-
|aac|agC|TTt|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat tgt gcg aga-3′
(H43.XABr2) 5′-ggtgtagtga-
|TCT|AGt|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
|aac|agC|TTt|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat tgt gcg aaa-3′
(H43.XAExt) 5′-ATAgTAgAcT gcAgTgTccT cAgcccTTAA gcTgTTcATc TgcAAgTAgA-
gAgTATTcTT AgAgTTgTcT cTAgATcAcT AcAcc-3′
!H43.XAExt is the reverse complement of
!5′-ggtgtagtga-
!  |TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
!  |aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat -3′
(H43.XAPcR) 5′-ggtgtagtga |TCT|AGA|gac|aac-3′
! XbaI and AflII sites in bridges are bunged
(H43.ABr1) 5′-ggtgtagtga-
|aac|agC|TTt|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat tgt gcg aga-3′
(H43.ABr2) 5′-ggtgtagtga-
|aac|aagC|TTt|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat tgt gcg aaa-3′
(H43.AExt)  5′- ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTTcAcTAcAcc-3′
!(H43.AExt) is the reverse complement of 5′-ggtgtagtga-
′  |aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat-3′
(H43.APCR)       5′-ggtgtagtga |aac|agC|TTA|AGg|gct|g-3′

[0154]

(FOKIact) 5′-cAcATccgTg TTgTT cAcggATgTg-3′
(VHEx881) 5′-AATAgTAgAc TgcAgTgTcc TcAgcccTTA AgcTgTTcAT cTgcAAgTAg-
AgAgTATTcT TAgAgTTgTc TcTAgAcTTA gTgAAgcg-3′
! note that VHEx881 is the reverse complement of the ON below
!         [RC] 5′-cgCttcacTaag-
!                 Scab........
!                 Synthetic 3-23 as in Table 206
!                 |TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
!                  XbaI...
!                 |aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|t-3′
!                      AflII...
(VHBA881)     5′-cgCttcacTaag-
|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgt gcg ag-3′
(VHBB881)      5′-cgCttcacTaag-
|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
1  |aac|agC|TTA|AGg|gct|gag|gac|acT|GcA|Gtc|tac|tat|tgt Acg ag-3′
(VH881PCR) 5′-cgCttcacTaag|TCT|AGA|gac|aac-3′

[0155]

TABLE 600
V3-23 VH framework with variegated codons shown
!
!            /////////                   17  18  19  20  21  22
!            //////////                    A   Q   P   A   M   A
             ///////// 5′-ctg tct gaa cG GCC cag ccG GCC atg gcc 29
             ///////// 3′-gac aga ctt gc cgg gtc ggc cgg tac cgg
!            /////////    Scab.........SfiI.............
!            ///////////////////////////////////////                         NgoMI...
!            /////////////////////////////////////////                                NcoI....
!
!            //////////////////////////// FR1(DP47/V3-23)---------------
!            //////////////////////////// 23  24  25  26  27  28  29  30
!            /////////////////////////////  E   V   Q   L   L   E   S   G
             //////////////////////////// gaa|gtt|CAA|TTG|tta|gag|tct|ggt| 53
!            //////////////////////////// ctt|caa|gtt|aac|aat|ctc|aga|cca|
!            ///////////////////////////////////        | MfeI  |
!
!            --------------FR1
!            31  32  33  34  35  36  37  38  39  40  41  42  43  44  45
!            G   G   L   V   Q   P   C   G   S   L   R   L   S   C   A
             |ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|                  cgt|ctt|tct|tgc|gct| 98
!            |ccg|cca|gaa|caa|gtc|gga|cca|cca|aga|aat|gca|gaa|aga|acg|cga|
!            Sites to be varied--->       ***     ***     ***
!            ----FR1---------------->|...CDR1................|---FR2------
!            46  47  48  49  50  51  52  53  54  55  56  57  58  59  60
!            A   S   G   F   T   F   S   S   Y   A   M   S   W   V   R
             |gct|TCC|GGA|ttc|act|ttc|                      tct|tCG|TAC|Gct|atg|tct|tgg|gtt|cgC| 143
!            |cga|agg|cct|aag|tga|aag                  |aga|agc|atg|cga|tac|aga|acc|caa|gcg|
!            | BspEI |                 | BsiWI|                     |BstXI.
!
!            Sites to be varies---> ***     *** ***
!            -------FR2--------------------------------->|...CDR2........
!            61  62  63  64  65  66  67  68  69  70  71  72  73  74  75
!            Q   A   P   G   K   G   L   E   W   V   S   A   I   S   G
             |CAa|gct|ccT|GG                                                            t|aaa|ggt|ttg|gag|tgg|gtt|tct|gct|atc|tct|ggt| 188
!            |gtt|cga|gga|cca|ttt|cca|aac|ctc|acc|caa|aga                  |cga|tag|aga|cca|
!            ...BstXI          |
!
!            ***     ***
!            .....CDR2............................................|---FR3---
!            76  77  78  79  80  81  82  83  84  85  86  87  88  89  90
!            S   G   G   S   T   Y   Y   A   D   S   V   K   G   R   F
             |tct|ggt|ggc|agt|act|tac|tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc| 233
!            |aga|cca|ccg|tca|tga|atg|ata|cga|ctg|agg|caa|ttt|cca|gcg|aag|
!
!            --------FR3--------------------------------------------------
!            91  92  93  94  95  96  97  98  99 100 101 102 103 104 105
!            T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M
             |act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| 278
!            |tga|tag|aga|tct|ctg|ttg|aga|ttc|tta|tga|gag|atg|aac|gtc|tac|
!            | XbaI  |
!
!            ---FR3----------------------------------------------------->|
!            106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
!            N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   K
             |aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa| 323
!            |ttg|tcg|aat|tcc|cga|ctc|ctg|tga                  |cgt|cag|atg|ata|acg|cga|ttt|
!            |AflII |               | PstI |
!
!            .......CDR3.................|----FR4-------------------------
!            121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
!            D   Y   E   G   T   G   Y   A   F   D   I   W   G   Q   G
             |gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gaC|ATA|TGg|ggt|c                  aa|ggt| 368
!            |ctg|ata|ctt|cca|tga|cca|ata|cga|aag|ctg|tat|acc|cca|gtt|cca|
!            | NdeI |
!
!            --------------FR4---------->|
!            136 137 138 139 140 141 142
!            T   N   V   T   V   S   S
             |act|atG|GTC|ACC|gtc|tct|agt-    389
!            |tga|tac|cag|tgg|cag|aga|tca                  -
!            | BstEII |
!
!            /////////////// 143 144 145 146 147 148 149 150 151 152
!            ////////////////  A   S   T   K   G   P   S   V   F   P
             /////////////// gcc tcc acc aaG GGC CCa tcg GTC TTC ccc-3′ 419
!            /////////////// cgg agg tgg tta ccg ggt agc cag aag ggg-5′
!            ////////////// ///////////////               Bsp120I.      BbsI...(2/2)
!            ////////////// ///////////////               ApaI....
!
(SFPRMET)    5′-ctg tct gaa cG GCC cag ccG-3′
(TOPFR1A)    5′-ctg tct gaa cG GCC cag ccG GCC atg gcc-
             gaa|gtt|CAA|TTG|tta|gag|tct|ggt|-
             |ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta-3′
(BOTR1B)     3′-caa|gtc|gga|cca|cca|aga|aat|gca|gaa|aga|acg|cga|-
             |cga|agg|cct|aag|tga|aag                  -5′ ! bottom strand
(BOTFR2)     3                  ′-acc|caa|gcg|-
             |gtt|cga|gga|cca|ttt|cca|aac|ctc|acc|caa|aga|-5′ ! bottom strand
(BOTFR3)     3                  ′-  a|cga|ctg|agg|caa|ttt|cca|gcg|aag|-
             |tga|tag|aga|tct|ctg|ttg|aga|ttc|tta|tga|gag|atg|aac|gtc|tac|-
             |ttg|tcg|aat|tcc|cga|ctc|ctg|tga                  -5′
(F06)        5′-gC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa|-
             |gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gac|ATA|TGg|ggt|c-3′
(BOTFR4)     3                  ′-cga|aag|ctg|tat|acc|cca|gtt|cca|-
             |tga|tac|cag|tgg|cag|aga|tca-
             cgg agg tgg ttc ccg ggt aga cag aag ggg-5′ ! bottom strand
(BOTPRCPRIM) 3′-gg ttc ccg ggt agc cag aag ggg-5′
!
!            CDR1 diversity
!
(ON-vgC1)    5′-|gct|TCC|GGA|tta|act|ttc|tct|<1>|TAC|<11>|atg|<1>|-
!            CDR1....................6859
             |tgg|gtt|cgc|CAa|gct|ccT|GG-3′
!
!<1> stands for an equimolar mix of (ADEFGHIKLMNPQRSTVWY); no C
!                              (this is not a sequence)
!            CDR2 diversity
!
(ON-vgC2)    5′-ggt|ttg|gag|tgg|gtt|tct|<2>|atc|<2>|<3>|-
!            CDR2............
             |tct|ggt|ggc|<1>|act|<1>|tat|gct|gac|tcc|gtt|aaa|gg-3′
!            CDR2................................................
!<1> is an equimolar mixture of {ADEFGHIKLMNPQRSTVWT}; no C
!<2> is an equimolar mixture of {YRWVGS}; no ACDEFHIKLMNPQT
!<3> is an equimolar mixture of {PS}; no ACDEFGHIKLMNQRTVWY

[0156]

TABLE 800
(new)
The following list of enzymes was taken from
http://rebase.neb.com/cgi-bin/asymmlist.
I have removed the enzymes that a) cut within the recognition, b)
cut on both sides of the recognition, or c) have fewer than 2
bases between recognition and closest cut site.
REBASE Enzymes
04/13/2001
Type II Restriction Enzymes with Asymmetric Recognition
Sequences:
Enzymes	Recognition Sequence	Isoschizomers	Suppliers
AarI	CACCTGCNNNN{circumflex over ( )}NNNN_	-	y
AceIII	CAGCTCNNNNNNN{circumflex over ( )}NNNN_	-	-
Bbr7I	GAAGACNNNNNNN{circumflex over ( )}NNNN_	-	-
BbvI	GCAGCNNNNNNNN{circumflex over ( )}NNNN_		y
BbvII	GAAGACNN{circumflex over ( )}NNNN_
Bce83I	CTTGAGNNNNNNNNNNNNNN_NN{circumflex over ( )}	-	-
BceAI	ACGGCNNNNNNNNNNNN{circumflex over ( )}NN_	-	y
BcefI	ACGGCNNNNNNNNNNNN{circumflex over ( )}N_	-	-
BciVI	GTATCCNNNNN_N{circumflex over ( )}	BfuI	y
BfiI	ACTGGGNNNN_N{circumflex over ( )}	BmrI	y
BinI	GGATCNNNN{circumflex over ( )}N_
BscAI	GCATCNNNN{circumflex over ( )}NN_	-	-
BseRI	GAGGAGNNNNNNNN_NN{circumflex over ( )}	-	y
BsmFI	GGGACNNNNNNNNNN{circumflex over ( )}NNNN_	BspLU11III	y
BspMI	ACCTGCNNNN{circumflex over ( )}NNNN_	Acc36I	y
EciI	GGCGGANNNNNNNNN_NN{circumflex over ( )}	-	y
Eco57I	CTGAAGNNNNNNNNNNNNNN_NN{circumflex over ( )}	BspKT5I	y
FauI	CCCGCNNNNNN_	BstFZ438I	y
FokI	GGATGNNNNNNNNN{circumflex over ( )}NNNN_	BstPZ418I	y
GsuI	CTGGAGNNNNNNNNNNNNNN_NN{circumflex over ( )}	-	y
HgaI	GACGCNNNNN{circumflex over ( )}NNNNN_	-	y
HphI	GGTGANNNNNNN_N{circumflex over ( )}	AsuHPI	y
MboII	GAAGANNNNNNN_N{circumflex over ( )}	-	y
MlyI	GAGTCNNNNN{circumflex over ( )}	SchI	y
MmeI	TCCRACNNNNNNNNNNNNNNNNNN_NN{circumflex over ( )}	-	-
MnlI	CCTCNNNNNN_N{circumflex over ( )}	-	y
PleI	GAGTCNNNN{circumflex over ( )}N_	PpsI	y
RleAI	CCCACANNNNNNNNN_NNN{circumflex over ( )}	-	-
SfaNI	GCATCNNNNN{circumflex over ( )}NNNN_	BspST5I	y
SspD5I	GGTGANNNNNNNN{circumflex over ( )}	-	-
Sth132I	CCCGNNNN{circumflex over ( )}NNNN_	-	-
StsI	GGATGNNNNNNNNNN{circumflex over ( )}NNNN	-	-
TaqII	GACCGANNNNNNNNN_NN{circumflex over ( )}, CACCCANNNNNNNNN_NN{circumflex over ( )}	-	-
Tth111II	CAARCANNNNNNNNN_NN{circumflex over ( )}	-	-
UbaPI	CGAACG	-	-

[0157] The notation is {circumflex over ( )} means cut the upper strand and _ means cut the lower strand. If the upper and lower strand are cut at the same place, then only {circumflex over ( )} appears.

TABLE 120
MALIA3, annotated
! MALIA3 9532 bases
!--------------------------------------------------------------------
	1	aat gct act act att agt aga att gat gcc acc ttt tca gct cgc gcc
!	gene ii continued
	49	cca aat gaa aat ata gct aaa cag gtt att gac cat ttg cga aat gta
	97	tct aat ggt caa act aaa tct act cgt tcg cag aat tgg gaa tca act
	145	gtt aca tgg aat gaa act tcc aga cac cgt act tta gtt gca tat tta
	193	aaa cat gtt gag cta cag cac cag att cag caa tta agc tct aag cca
	241	tcc gca aaa atg acc tct tat caa aag gag caa tta aag gta ctc tct
	289	aat cct gac ctg ttg gag ttt gct tcc ggt ctg gtt cgc ttt gaa gct
	337	cga att aaa acg cga tat ttg aag tct ttc ggg ctt cct ctt aat ctt
	385	ttt gat gca atc cgc ttt gct tct gac tat aat agt cag ggt aaa gac
	433	ctg att ttt gat tta tgg tca ttc tcg ttt tct gaa ctg ttt aaa gca
	481	ttt gag ggg gat tca ATG aat att tat gac gat tcc gca gta ttg gac
!		RBS?......      Start gene x, ii continues
	529	gct atc cag tct aaa cat ttt act att acc ccc tct ggc aaa act tct
	577	ttt gca aaa gcc tct cgc tat ttt ggt ttt tat cgt cgt ctg gta aac
	625	gag ggt tat gat agt gtt gct ctt act atg cct cgt aat tcc ttt tgg
	673	cgt tat gta tct gca tta gtt gaa tgt ggt att cct aaa tct caa ctg
	721	atg aat ctt tct acc tgt aat aat gtt gtt ccg tta gtt cgt ttt att
	769	aac gta gat ttt tct tcc caa cgt cct gac tgg tat aat gag cca gtt
	817	ctt aaa atc gca TAA
!		End X & II
	832	ggtaattca ca
!
!		M1              E5                 Q10                 T15
	843	ATG att aaa gtt gaa att aaa cca tct caa gcc caa ttt act act cgt
!		Start gene V
!		S17         S20                 P25                 E30
	891	tct ggt gtt tct cgt cag ggc aag cct tat tca ctg aat gag cag ctt
!
!		V35                 E40                 V45
	939	tgt tac gtt gat ttg ggt aat gaa tat ccg gtt ctt gtc aag att act
!
!		D50                 A55                 L60
	987	ctt gat gaa ggt cag cca gcc tat gcg cct ggt cTG TAC Acc gtt cat
!		BsrGI...
!		L65                 V70                 S75                 R80
	1035	ctg tcc tct ttc aaa gtt ggt cag ttc ggt tcc ctt atg att gac cgt
!
!		P85     K87 end of V
	1083	ctg cgc ctc gtt ccg gct aag TAA C
!
	1108	ATG gag cag gtc gcg gat ttc gac aca att tat cag gcg atg
!		Start gene VII
!
	1150	ata caa atc tcc gtt gta ctt tgt ttc gcg att ggt ata atc
!
!		VII and IX overlap.
!		..... S2  V3  L4  V5                 S10
	1192	gct ggg ggt caa agA TGA gt gtt tta gtg tat tct ttc gcc tct ttc gtt
!		End VII
!		|start IX
!		L13     W15                 G20                 T25             E29
	1242	tta ggt tgg tgc ctt cgt agt ggc att acg tat ttt acc cgt tta atg gaa
!
	1293	act tcc tc
!
!		.... stop of IX, IX and VIII overlap by four bases
	1301	ATG aaa aag tct tta gtc ctc aaa gcc tct gta gcc gtt gct acc ctc
!		Start signal sequence of viii.
!
	1349	gtt ccg atg ctg tct ttc gct gct gag ggt gac gat ccc gca aaa gcg
!		mature VIII --->
	1397	gcc ttt aac tcc ctg caa gcc tca gcg acc gaa tat atc ggt tat gcg
	1445	tgg gcg atg gtt gtt gtc att
	1466	gtc ggc gca act atc ggt atc aag ctg ttt aag
	1499	aaa ttc acc tcg aaa gca ! 1515
!		...........  -35  ..
!
	1517	agc tga taaaccgat acaattaaag gctccttttg
!		..... -10   ...
	1552	gagccttttt ttttGGAGAt ttt ? S.D. underlined
!
!		<------ III signal sequence -------------------------->
!		M   K   K   L   L   F   A   I   P   L   V
	1575	caac GTG aaa aaa tta tta ttc gca att cct tta gtt ! 1611
!
!		V   P   F   Y   S   H   S   A   Q
	1612	gtt cct ttc tat tct cac aGT gcA Cag tCT
!		ApaLI...
!
	1642	GTC GTG ACG CAG CCG CCC TCA GTG TCT GGG GCC CCA GGG GAG
		AGG GTC ACC ATC TCC TGC ACT GGG AGC AGC TCC AAC ATC GGG GCA
!		BstEII...
	1729	GGT TAT GAT GTA CAC TGG TAC CAG CAG CTT CCA GGA ACA GCC CCC AAA
	1777	CTC CTC ATC TAT GGT AAC AGC AAT CGG CCC TCA GGG GTC CCT GAC CGA
	1825	TTC TCT GGC TCC AAG TCT GGC ACC TCA GCC TCC CTG GCC ATC ACT
	1870	GGG CTC CAG GCT GAG GAT GAG GCT GAT TAT
	1900	TAG TGC CAG TCC TAT GAC AGC AGC CTG AGT
	1930	GGC CTT TAT GTC TTC GGA ACT GGG ACC AAG GTC ACC GTC
!		BstEII...
	1969	CTA GGT CAG CCC AAG GCC AAC CCC ACT GTC ACT
	2002	CTG TTC CCG CCC TCC TCT GAG GAG CTC CAA GCC AAC AAG GCC ACA CTA
	2050	GTG TGT CTG ATC AGT GAC TTC TAC CCG GGA GCT GTG ACA GTG GCC TGG
	2098	AAG GCA GAT AGC AGC CCC GTC AAG GCG GGA GTG GAG ACC ACC ACA CCC
	2146	TCC AAA CAA AGC AAC AAC AAG TAC GCG GCC AGC AGC TAT CTG AGC CTG
	2194	ACG CCT GAG CAG TGG AAG TCC CAC AGA AGC TAC AGC TGC CAG GTC ACG
	2242	CAT GAA GGG AGC ACC GTG GAG AAG ACA GTG GCC CCT ACA GAA TGT TCA
	2290	TAA TAA ACCG CCTCCACCGG GCGCGCCAAT TCTATTTCAA GGAGACAGTG ATA
!		AscI.....
!
!		PelB signal---------------------------------------------->
!		M   K   Y   L   L   P   T   A   A   A   C   L   L   L   L
	2343	ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC
!
!		16  17  18  19  20      21  22
!		A   A   Q   P   A       M   A
	2388	gcG GCC cag ccG GCC     atg gcc
!		SfiI.............
!		NgoMI...(1/2)
!		NcoI.........
!
!		FR1 (DP47/V3-23)--------------
!		23  24  25  26  27  28  29  30
!		E   V   Q   L   L   E   S   G
	2409	gaa|gtt|CAA|TTG|tta|gag|tct|ggt|
!		|MfeI  |
!
!		--------------FR1--------------------------------------------
!		31  32  33  34  35  36  37  38  39  40  41  42  43  44  45
!		G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A
	2433	|ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct|
!
!		----FR1---------------->|...CDR1................|---FR2------
!		46  47  48  49  50  51  52  53  54  55  56  57  58  59  60
		A   S   G   F   T   F   S   S   Y   A   M   S   W   V   R
	2478	|gct|TCC|GGA|ttc|act|ttc|tct|tCG|TAC|Gct|atg|tct|tgg|gtt|cgC|
!		|BspEI |                 |BsiWI|                     |BstXI
!
!		-------FR2-------------------------------->|...CDR2.........
!		61  62  63  64  65  66  67  68  69  70  71  72  73  74  75
!		Q   A   P   G   K   G   L   E   W   V   S   A   I   S   G
	2523	|CAa|gct|ccT|GGt|aaa|ggt|ttg|gag|tgg|gtt|tct|gct|atc|tct|ggt|
!		...BstXI          |
!
!		.....CDR2............................................|---FR3---
!		76  77  78  79  80  81  82  83  84  85  86  87  88  89  90
!		S   C   C   S   T   Y   Y   A   D   S   V   K   G   R   F
!	2568	|tct|ggt|ggc|agt|act|tac|tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc|
!
!
!		--------FR3--------------------------------------------------
!		91  92  93  94  95  96  97  98  99 100 101 102 103 104 105
!		T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M
	2613	|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|
!		|XbaI  |
!
!		---FR3----------------------------------------------------->|
!		106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
!		N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   K
	2658	|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa|
!		|AflII |               |PstI |
!
!		.......CDR3.................|----FR4-------------------------
!		121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
!		D   Y   E   G   T   G   Y   A   F   D   I   W   G   Q   G
	2703	|gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gaC|ATA|TGg|ggt|caa|ggt|
!		|NdeI |(1/4)
!
!		--------------FR4---------->|
!		136 137 138 139 140 141 142
!		T   M   V   T   V   S   S
	2748	|act|atG|GTC|ACC|gtc|tct|agt
!		|BstEII |
!	From BstEII onwards, pV323 is same as pCES1, except as noted.
!	BstEII sites may occur in light chains; not likely to be unique in final
!	vector.
!
!		143 144 145 146 147 148 149 150 151 152
!		A   S   T   K   G   P   S   V   F   P
	2769	gcc tcc acc aaG GGC CCa tcg GTC TTC ccc
!		Bsp120I.      BbsI...(2/2)
!		ApaI....
!
!		153 154 155 156 157 158 159 160 161 162 163 164 165 166 167
!		L   A   P   S   S   K   S   T   S   G   G   T   A   A   L
	2799	ctg gca ccC TCC TCc aag agc acc tct ggg ggc aca gcg gcc ctg
!		BseRI...(2/2)
!
!		168 169 170 171 172 173 174 175 176 177 178 179 180 181 182
!		G   C   L   V   K   D   Y   F   P   E   P   V   T   V   S
	2844	ggc tgc ctg GTC AAG GAC TAC TTC CCc gaA CCG GTg acg gtg tcg
!		AgeI....
!
!		183 184 185 186 187 188 189 190 191 192 193 194 195 196 197
!		W   N   S   G   A   L   T   S   G   V   H   T   F   P   A
	2889	tgg aac tca GGC GCC ctg acc agc ggc gtc cac acc ttc ccg gct
!		KasI...(1/4)
!
!		198 199 200 201 202 203 204 205 206 207 208 209 210 211 212
!		V   L   Q   S   S   G   L   Y   S   I   S   S   V   V   T
	2934	gtc cta cag tCt agc GGa ctc tac tcc ctc agc agc gta gtg acc
!		(Bsu36I...)(knocked out)
!
!		213 214 215 216 217 218 219 220 221 222 223 224 225 226 227
!		V   P   S   S   S   L   G   T   Q   T   Y   I   C   N   V
	2979	gtg ccC tCt tct agc tTG Ggc acc cag acc tac atc tgc aac gtg
!		(BstXI...........)N.B. destruction of BstXI & BpmI sites.
!
!		228 229 230 231 232 233 234 235 236 237 238 239 240 241 242
!		N   H   K   P   S   N   T   K   V   D   K   K   V   E   P
	3024	aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc
!
!		243 244 245
!		K   S   C   A   A   A   H   H   H   H   H   H   S   A
	3069	aaa tct tgt GCG GCC GCt cat cac cac cat cat cac tct gct
!		NotI......
!
!		E   Q   K   L   I   S   E   E   D   L   N   G   A   A
	3111	gaa caa aaa ctc atc tca gaa gag gat ctg aat ggt gcc gca
!
!
!		D   I   N   D   D   R   M     A   S    G   A
	3153	GAT ATC aac gat gat cgt atg   gct AGC  ggc gcc
!		rEK cleavage site..........   NheI...  KasI...
!		EcoRV..
!
!	Domain	1 ---------------------------------------------------------
!		A   E   T   V   E   S   C   L   A
!	3183	gct gaa act gtt gaa agt tgt tta gca
!
!
!		K   P   H   T   E   I   S   F
	3210	aaa ccc cat aca gaa aat tca ttt
!
!		T   N   V   W   K   D   D   K   T
	3234	aCT AAC GTC TGG AAA GAC GAC AAA ACt
!
!		L   D   R   Y   A   N   Y   E   G   C   L   W   N   A   T   G   V
	3261	tta gat cgt tac gct aac tat gag ggt tgt ctg tgG AAT GCt aca ggc gtt
!		BsmI
!
!		V   V   C   T   G   D   E   T   Q   C   Y   G   T   W   V   P   I
	3312	gta gtt tgt act ggt GAC GAA ACT GAG TGT TAC GGT ACA TGG GTT cct att
!
!		G   L   A   I   P   E   N
	3363	ggg ctt gct atc cct gaa aat
!
!	L1 linker	-------------------------------------
!		E   G   G   G   S   E   G   G   G   S
	3384	gag ggt ggt ggc tct gag ggt ggc ggt tct
!
!		E   G   G   G   S   E   G   G   G   T
	3414	gag ggt ggc ggt tct gag ggt ggc ggt act
!
!	Domain	2 ---------------------------------------
	3444	aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat atc aac
	3495	cct ctc gac ggc act tat ccg cct ggt act gag caa aac ccc gct aat cct
	3546	aat cct tct ctt GAG GAG tct cag cct ctt aat act ttc atg ttt cag aat
!		BseRI
	3597	aat agg ttc cga aat agg cag ggg gca tta act gtt tat acg ggc act
	3645	gtt act caa ggc act gac ccc gtt aaa act tat tac cag tac act cct
	3693	gta tca tca aaa gcc atg tat gac gct tac tgg aac ggt aaa ttC AGA
!		AlwNI
	3741	GAC TGc gct ttc cat tct ggc ttt aat gaa gat cca ttc gtt tgt gaa
!		AlwNI
	3789	tat caa ggc caa tcg tct gac ctg cct caa cct cct gtc aat gct
!
	3834	ggc ggc ggc tct
!	start	L2 --------------------------------------------------------------
	3846	ggt ggt ggt tct
	3858	ggt ggc ggc tct
	3870	gag ggt ggt ggc tct gag ggt ggc ggt tct
	3900	gag ggt ggc ggc tct gag gga ggc ggt tcc
	3930	ggt ggt ggc tct ggt    ! end L2
!
!	Domain	3 ---------------------------------------------------------------
!		S   G   D   F   D   Y   E   K   M   A   N   A   N   K   G   A
	3945	tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat aag ggg gct
!
!		M   T   E   N   A   D   E   N   A   L   Q   S   D   A   K   G
	3993	atg acc gaa aat gcc gat gaa aac gag cta cag tct gac gct aaa ggc
!
!		K   L   D   S   V   A   T   D   Y   G   A   A   I   D   G   F
	4041	aaa ctt gat tct gtc gct act gat tac ggt gat gct atc gat ggt ttc
!
!		I   G   D   V   S   G   L   A   N   G   N   G   A   T   G   D
	4089	att ggt gac gtt tcc ggc ctt gat aat ggt aat ggt gct act ggt gat
!
!		F   A   G   S   N   S   Q   M   A   Q   V   G   D   G   D   N
	4137	ttt gat ggc tat aat tcc caa atg gct caa gtc ggt gac ggt gat aat
!
!		S   P   L   M   N   N   F   R   Q   Y   L   P   S   L   P   Q
	4185	tca cat tta atg aat aat ttc cgt caa tat tta cct tcc atc cat caa
!
!		S   V   E   C   R   P   F   V   F   S   A   G   K   P   Y   E
	4233	tcg gtt gaa tgt cgc cct ttt gtc ttt aga gct ggt aaa cca tat gaa
!
!		F   S   I   D   C   D   K   I   N   L   F   R
	4281	ttt tct att gat tgt gac aaa ata aac tta tta cgt
!		End Domain 3
!
!		G   V   F   A   F   L   L   Y   V   A   T   F   M   Y   V  F140
	4317	ggt gtc ttt gcg ttt ctt tta tat gtt gcc aac ttt atg tat gta ttt
!		start transmembrane segment
!
!		S   T   F   A   N   I   L
	4365	tat acg ttt gct aac ata ctg
!
!		R   N   K   E   S
	4386	cgt aat aag gag tct TAA ! stop of iii
!		Intracellular anchor.
!
!		M1  P2  V   L  L5   G   I   P   L  L10  L   R   F   L  G15
	4404	tc ATG cca gtt ctt ttg ggt att ccg tta tta ttg cgt ttc ctc ggt
!		Start VI
!
	4451	ttc ctt ctg gta act ttg ttc ggc tat ctg ctt act ttt ctt aaa aag
	4499	ggc ttc ggt aag ata gct att gct att tca ttg ttt ctt gct ctt att
	4547	att ggg ctt aac tca att ctt gtg ggt tat ctc tct gat att agc gct
	4595	caa tta ccc tct gac ttt gtt cag ggt gtt cag tta att ctc ccg tct
	4643	aat gcg ctt ccc tgt ttt tat gtt att ctc tct gta aag gct gct att
	4691	ttc att ttt gac gtt aaa caa aaa atc gtt tct tat ttg gat tgg gat
!
!		M1  A2  V3      F5                 L10         G13
	4739	aaa TAA t ATG gct gtt tat ttt gta act ggc aaa tta ggc tct gga
!		end VI   Start gene I
!
!		14  15  16  17  18  19  20  21  22  23  24  25  26  27  28
!		K   T   L   V   S   V   G   K   I   Q   D   K   I   V   A
	4785	aag acg ctc gtt agc gtt ggt aag att cag gat aaa att gta gct
!
!		29  30  31  32  33  34  35  36  37  38  39  40  41  42  43
!		G   C   K   I   A   T   N   L   D   L   R   L   Q   N   L
	4830	ggg tgc aaa ata gca act aat ctt gat tta agg ctt caa aac ctc
!
!		44  45  46  47  48  49  50  51  52  53  54  55  56  57  58
!		P   Q   V   G   R   F   A   K   T   P   I   V   L   R   I
	4875	ccg caa gtc ggg agg ttc gct aaa acg cct cgc gtt ctt aga ata
!
!		59  60  61  62  63  64  65  66  67  68  69  70  71  72  73
!		P   D   K   P   S   I   S   D   L   L   A   I   G   R   G
	4920	ccg gat aag cct tct ata tct gat ttg ctt gct att ggg cgc ggt
!
!		74  75  76  77  78  79  80  81  82  83  84  85  86  87  88
!		N   D   S   Y   D   E   N   K   N   G   L   L   V   L   D
	4965	aat gat tcc tac gat gaa aat aaa aac ggc ttg ctt gtt ctc gat
!
!		89  90  91  92  93  94  95  96  97  98  99 100 101 102 103
!		E   C   G   T   W   F   N   T   R   S   W   N   D   K   E
	5010	gag tgc ggt act tgg ttt aat acc cgt tct tgg aat gat aag gaa
!
!		104 105 106 107 108 109 110 111 112 113 114 115 116 117 118
!		R   Q   P   I   I   D   W   F   L   H   A   R   K   L   G
	5055	aga cag ccg att att gat tgg ttt cta cat gct cgt aaa tta gga
!
!		119 120 121 122 123 124 125 126 127 128 129 130 131 132 133
!		W   D   I   I   F   L   V   Q   D   L   S   I   V   D   K
	5100	tgg gat att att ttt ctt gtt cag gac tta tct att gtt gat aaa
!
!		134 135 136 137 138 139 140 141 142 143 144 145 146 147 148
!		Q   A   R   S   A   L   A   E   H   V   V   Y   C   R   R
	5145	cag gcg cgt tct gca tta gct gaa cat gtt gtt tat tgt cgt cgt
!
!		149 150 151 152 153 154 155 156 157 158 159 160 161 162 163
!		L   D   R   I   T   L   P   F   V   G   T   L   Y   S   L
	5190	ctg gac aga att act tta cct ttt gtc ggt act tta tat tct ctt
!
!		164 165 166 167 168 169 170 171 172 173 174 175 176 177 178
!		I   T   G   S   K   M   P   L   P   K   L   H   V   C   V
	5235	att act ggc tcg aaa atg cct ctg cct aaa tta cat gtt ggc gtt
!
!		179 180 181 182 183 184 185 186 187 188 189 190 191 192 193
!		V   K   Y   G   D   S   Q   L   S   P   T   V   E   R   W
	5280	gtt aaa tat ggc gat tct caa tta agc cct act gtt gag cgt tgg
!
!		194 195 196 197 198 199 200 201 202 203 204 205 206 207 208
!		L   Y   T   C   K   N   L   Y   N   A   Y   D   T   K   Q
	5325	ctt tat act ggt aag aat ttg tat aac gca tat gat act aaa cag
!
!		209 210 211 212 213 214 215 216 217 218 219 220 221 222 223
!		A   F   S   S   N   Y   D   S   G   V   Y   S   Y   L   T
	5370	gct ttt tct agt aat tat gat tcc ggt gtt tat tct tat tta acg
!
!		224 225 226 227 228 229 230 231 232 233 234 235 236 237 238
!		P   Y   L   S   H   G   R   Y   F   K   P   L   N   L   G
	5415	cct tat tta tca cac ggt cgg tat ttc aaa cca tta aat tta ggt
!
!		239 240 241 242 243 244 245 246 247 248 249 250 251 252 253
!		Q   K   M   K   L   T   K   I   Y   L   K   K   F   S   R
	5460	cag aag atg aaa tta act aaa ata tat ttg aaa aag ttt tct cgc
!
!		254 255 256 257 258 259 260 261 262 263 264 265 266 267 268
!		V   L   C   L   A   I   G   F   A   S   A   F   T   Y   S
	5505	gtt ctt tgt ctt gcg att gga ttt gca tca gca ttt aca tat agt
!
!		269 270 271 272 273 274 275 276 277 278 279 280 281 282 283
!		Y   I   T   Q   P   K   P   E   V   K   K   V   V   S   Q
	5550	tat ata acc caa cct aag ccg gag gtt aaa aag gta gtc tct cag
!
!		284 285 286 287 288 289 290 291 292 293 294 295 296 297 298
!		T   Y   D   F   D   K   F   T   I   D   S   S   Q   R   L
	5595	acc tat gat ttt gat aaa ttc act att gac tct tct cag cgt ctt
!
!		299 300 301 302 303 304 305 306 307 308 309 310 311 312 313
!		N   L   S   Y   R   Y   V   F   K   D   S   K   G   K   L
	5640	aat cta agc tat cgc tat gtt ttc aag gat tct aag gga aaa TTA
!		PacI
!
!		314 315 316 317 318 319 320 321 322 323 324 325 326 327 328
!		I   N   S   D   D   L   Q   K   Q   G   Y   S   L   T   Y
	5685	ATT AAt agc gac gat tta cag aag caa ggt tat tca ctc aca tat
!		PacI
!
!		329 330 331 332 333 334 335 336 337 338 339 340 341 342 343
!		i  I   D   L   C   T   V   S   I   K   K   G   N   S   N   E
!		iv                                                       Ml  K
	5730	att gat tta tgt act gtt tcc att aaa aaa ggt aat tca aAT Gaa
!		Start IV
!
!		344 345 346 347 348 349
!		i    I   V   K   C   N   .End of I
!		iv    L3  L   N5  V   I7  N    F  V10
	5775	att gtt aaa tgt aat TAA T TTT GTT
!	IV continued.....
	5800	ttc ttg atg ttt gtt tca tca tct tct ttt gct cag gta att gaa atg
	5848	aat aat tcg cct ctg cgc gat ttt gta act tgg tat tca aag caa tca
	5896	ggc gaa tcc gtt att gtt tct ccc gat gta aaa ggt act gtt act gta
	5944	tat tca tct gac gtt aaa cct gaa aat cta cgc aat ttc ttt att tct
	5992	gtt tta cgt gct aat aat ttt gat atg gtt ggt tca att cct tcc ata
	6040	att cag aag tat aat cca aac aat cag gat tat att gat gaa ttg cca
	6088	tca tct gat aat cag gaa tat gat gat aat tcc gct cct tct ggt ggt
	6136	ttc ttt gtt ccg caa aat gat aat gtt act caa act ttt aaa att aat
	6184	aac gtt cgg gca aag gat tta ata cga gtt gtc gaa ttg ttt gta aag
	6232	tct aat act tct aaa tcc tca aat gta tta tct att gac ggc tct aat
	6280	cta tta gtt gtt TCT gca cct aaa gat att tta gat aac ctt cct caa
!		ApaLI removed
	6328	ttc ctt tct act gtt gat ttg cca act gac cag ata ttg att gag ggt
	6376	ttg ata ttt gag gtt cag caa ggt gat gct tta gat ttt tca ttt gct
	6424	gct ggc tct cag cgt ggc act gtt gca ggc ggt gtt aat act gac cgc
	6472	ctc acc tct gtt tta tct tct gct ggt ggt tcg ttc ggt att ttt aat
	6520	ggc gat gtt tta ggg cta tca gtt cgc gca tta aag act aat agc cat
	6568	tca aaa ata ttg tct gtg cca cgt att ctt acg ctt tca ggt cag aag
	6616	ggt tct atc tct gtT GGC CAg aat gtc cct ttt att act ggt cgt gtg
!		MscI
	6664	act ggt gaa tct gec aat gta aat aat cca ttt cag acg att gag cgt
	6712	caa aat gta ggt att tcc atg agc gtt ttt cct gtt gca atg gct ggc
	6760	ggt aat att gtt ctg gat att acc agc aag gcc gat agt ttg agt tct
	6808	tct act cag gca agt gat gtt att act aat caa aga agt att gct aca
	6856	acg gtt aat ttg cgt gat gga cag act ctt tta ctc ggt ggc ctc act
	6904	gat tat aaa aac act tct caa gat tct ggc gta ccg ttc ctg tct aaa
	6952	atc cct tta atc ggc ctc ctg ttt agc tcc cgc tct gat tcc aac gag
	7000	gaa agc acg tta tac gtg ctc gtc aaa gca acc ata gta cgc gcc ctg
	7048	TAG cggcgcatt
!		End IV
	7060	aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
	7120	gcccgctcct ttcgctttct tccgttcctt tctcgccacg ttcGCCGGCt ttccccgtca
!		NgoMI
	7180	agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
	7240	caaaaaactt gatttgggtg atggttCACG TAGTGggcca tcgccctgat agacggtttt
!		DraIII
	7300	tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc aaactggaac
!		DrdI
	7360	aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga
	7420	accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgaaa
	7480	ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCTCact ggtgaaaaga
!		PvuII.      BsmBI.
	7540	aaaaccaccc tGGATCC  AAGCTT
!		BamHI   HindIII (1/2)
!		Insert carrying bla gene
	7563	gcaggtg gcacttttcg gggaaatgtg cgcggaaccc
	7600	ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct
!		BciVI
	7660	gataaatgct tcaataatat tgaaaaAGGA AGAgt
!		RBS.?...
!		Start bla gene
	7695	ATG agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca ttt
	7746	tgc ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa gta aaa gat gct
	7797	gaa gat cag ttg ggC gCA CGA Gtg ggt tac atc gaa ctg gat ctc aac agc
!		BssSI...
!		ApaLI removed
	7848	ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc
	7899	act ttt aaa gtt ctg cta tgt cat aca cta tta tcc cgt att gac gcc ggg
	7950	caa gaG CAA CTC GGT CGc cgg gcg cgg tat tct cag aat gac ttg gtt gAG
!		BcgI                                                       ScaI
	8001	TAC Tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa
!		ScaI
	8052	tta tgc agt gct gcc ata aca atg agt gat aac act gcg gcc aac tta ctt
	8103	ctg aca aCG ATC Gga gga ccg aag gag cta acc gct ttt ttg cac aac atg
!		PvuI
	8154	ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc
	8205	ata cca aac gac gag cgt gac acc acg atg cct gta gca atg cca aca acg
	8256	tTG CGC Aaa cta tta act ggc gaa cta ctt act cta gct tcc cgg caa caa
!		FspI....
	8307	tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt ctg cgc tcg
	8358	GCC ctt ccG GCt ggc tgg ttt att gct gat aaa tct gga gcc ggt gag cgt
!		BglI
	8409	gGG TCT Cgc ggt atc att gca gca ctg ggg cca gat ggt aag ccc tcc cgt
!		BsaI
	8460	atc gta gtt atc tac acG ACg ggg aGT Cag gca act atg gat gaa cga aat
!		AhdI
	8511	aga cag atc gct gag ata ggt gcc tca ctg att aag cat tgg TAA ctgt
!		stop
	8560	cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
	8620	ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
	8680	cgttccactg tacgtaagac cccc
	8704	AAGCTT   GTCGAC tgaa tggcgaatgg cgctttgcct
!		HindIII  SalI..
!		(2/2)    HincII
	8740	ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt
!
	8790	CCTGAGG
!		Bsu36I
	8797	ccgat actgtcgtcg tcccctcaaa ctggcagatg
	8832	cacggttacg atgcgcccat ctacaccaac gtaacctatc ccattacggt caatccgccg
	8892	tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc
	8952	tggctacagg aaggccagac gcgaattatt tttgatggcg ttcctattgg ttaaaaaatg
	9012	agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaATTTAAA
!		SwaI...
	9072	Tatttgctta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat
!		RBS?
	9131	ATG att gac atg cta gtt tta cga tta ccg ttc atc gat tct ctt gtt tgc
!		Start gene II
	9182	tcc aga ctc tca ggc aat gac ctg ata gcc ttt gtA GAT CTc tca aaa ata
!		BglII...
	9233	gct acc ctc tcc ggc atg aat tta tca gct aga acg gtt gaa tat cat att
	9284	gat ggt gat ttg act gtc tcc ggc ctt tct cac cct ttt gaa tct tta cct
	9335	aca cat tac tca ggc att gca ttt aaa ata tat gag ggt tct aaa aat ttt
	9386	tat cct tgc gtt gaa ata aag gct tct ccc gca aaa gta tta cag ggt cat
	9437	aat gtt ttt ggt aca acc gat tta gct tta tgc tct gag gct tta ttg ctt
	9488	aat ttt gct aat tct ttg cct tgc ctg tat gat tta ttg gat gtt ! 9532
!	gene II continues

[0158]

TABLE 120B
Sequence of MALIA3, condensed
LOCUS
ORIGIN MALIA3 9532 CIRCULAR
1    AATGCTACTA CTATTAGTAG AATTGATGCC ACCTTTTCAG CTCGCGCCCC AAATGAAAAT
61   ATAGCTAAAC AGGTTATTGA CCATTTGCGA AATGTATCTA ATGCTCAAAC TAAATCTACT
121  CGTTCGCAGA ATTGGGAATC AACTGTTACA TGGAATCAAA CTTCCAGACA CCGTACTTTA
181  GTTGCATATT TAAAACATGT TGAGCTACAG CACCAGATTC AGCAATTAAG CTCTAAGCCA
241  TCCGCAAAAA TGACCTCTTA TCAAAAGGAG CAATTAAAGG TACTCTCTAA TCCTGACCTG
301  TTGGAGTTTG CTTCCGGTCT GGTTCGCTTT GAAGCTCGAA TTAAAACGCG ATATTTGAAG
361  TCTTTCGGGC TTCCTCTTAA TCTTTTTGAT GCAATCCGCT TTGCTTCTGA CTATAATAGT
421  CAGGGTAAAG ACCTGATTTT TGATTTATGG TCATTCTCGT TTTCTGAACT GTTTAAAGCA
481  TTTGAGGGGG ATTCAATGAA TATTTATGAC GATTCCGCAG TATTGGACGC TATCCAGTCT
541  AAACATTTTA CTATTACCCC CTCTGGCAAA ACTTCTTTTG CAAAAGCCTC TCGCTATTTT
601  GGTTTTTATC GTCGTCTGGT AAACGAGGGT TATGATAGTG TTGCTCTTAC TATGCCTCGT
661  AATTCCTTTT GGCGTTATGT ATCTGCATTA GTTGAATGTG GTATTCCTAA ATCTCAACTG
721  ATGAATCTTT CTACCTGTAA TAATGTTGTT CCGTTAGTTC GTTTTATTAA CGTAGATTTT
781  TCTTCCCAAC GTCCTGACTG GTATAATGAG CCAGTTCTTA AAATCGCATA AGGTAATTCA
841  CAATGATTAA AGTTGAAATT AAACCATCTC AAGCCCAATT TACTACTCGT TCTGGTGTTT
901  CTCGTCAGGG CAAGCCTTAT TCACTGAATG AGCAGCTTTG TTACGTTGAT TTGGGTAATG
961  AATATCCCGT TCTTGTCAAG ATTACTCTTG ATGAAGGTCA GCCAGCCTAT GCGCCTGGTC
1021 TGTACACCGT TCATCTGTCC TCTTTCAAAG TTGGTCAGTT CGGTTCCCTT ATGATTGACC
1081 GTCTGCGCCT CGTTCCGGCT AAGTAACATG GAGCAGGTCG CGGATTTCGA CACAATTTAT
1141 CAGGCGATGA TACAAATCTC CGTTGTACTT TGTTTCGCGC TTGGTATAAT CGCTGGGGGT
1201 CAAAGATGAG TGTTTTAGTG TATTCTTTCG CCTCTTTCGT TTTAGGTTGG TGCCTTCGTA
1261 GTGGCATTAC GTATTTTACC CGTTTAATGG AAACTTCCTC ATGAAAAAGT CTTTAGTCCT
1321 CAAAGCCTCT GTAGCCGTTG CTACCCTCGT TCCGATGCTG TCTTTCGCTG CTGAGGGTGA
1381 CGATCCCGCA AAAGCGGCCT TTAACTCCCT GCAAGCCTCA GCGACCGAAT ATATCGGTTA
1441 TGCGTGGGCG ATGGTTGTTG TCATTCTCGG CGCAACTATC GGTATCAAGC TGTTTAAGAA
1501 ATTCACCTCG AAAGCAAGCT GATAAACCGA TACAATTAAA GGCTCCTTTT GGAGCCTTTT
1561 TTTTTGGAGA TTTTCAACGT GAAAAAATTA TTATTCGCAA TTCCTTTAGT TGTTCCTTTC
1621 TATTCTCACA GTGCACAGTC TGTCGTGACG CAGCCGCCCT CAGTGTCTGG GGCCCCAGGG
1681 CAGAGGGTCA CCATCTCCTG CACTGGGAGC AGCTCCAACA TCGGGGCAGG TTATGATGTA
1741 CACTGGTACC AGCAGCTTCC AGGAACAGCC CCCAAACTCC TCATCTATGG TAACAGCAAT
1801 CGGCCCTCAG GGGTCCCTGA CCGATTCTCT GGCTCCAAGT CTGGCACCTC AGCCTCCCTG
1861 GCCATCACTG GGCTCCAGGC TGAGGATGAG GCTGATTATT ACTGCCAGTC CTATGACAGC
1921 AGCCTGAGTG GCCTTTATGT CTTCGGAACT GGGACCAAGG TCACCGTCCT AGGTCAGCCC
1981 AAGGCCAACC CCACTGTCAC TCTGTTCCCG CCCTCCTCTG AGGAGCTCCA AGCCAACAAG
2041 GCCACACTAG TGTGTCTGAT CAGTGACTTC TACCCGGGAG CTGTGACAGT GGCCTGGAAG
2101 GCAGATAGCA GCCCCGTCAA GGCGGGAGTG GAGACCACCA CACCCTCCAA ACAAAGCAAC
2161 AACAAGTACG CGGCCAGCAG CTATCTGAGC CTGACGCCTG AGCAGTGGAA GTCCCACAGA
2221 AGCTACAGCT GCCAGGTCAC GCATGAAGGG AGCACCGTGG AGAAGACAGT GGCCCCTACA
2281 GAATGTTCAT AATAAACCGC CTCCACCGGG CGCGCCAATT CTATTTCAAG GAGACAGTCA
2341 TAATGAAATA CCTATTGCCT ACGGCAGCCG CTGGATTGTT ATTACTCGCG GCCCAGCCGG
2401 CCATGGCCGA AGTTCAATTG TTAGAGTCTG GTGGCGGTCT TGTTCAGCCT GGTGGTTCTT
2461 TACGTCTTTC TTGCGCTGCT TCCGGATTCA CTTTCTCTTC GTACGCTATG TCTTGGGTTC
2521 GCCAAGCTCC TGGTAAAGGT TTGGAGTGGG TTTCTGCTAT CTCTGGTTCT GGTGGCAGTA
2581 CTTACTATGC TGACTCCGTT AAAGGTCGCT TCACTATCTC TAGAGACAAC TCTAAGAATA
2641 CTCTCTACTT GCAGATGAAC AGCTTAAGGG CTGAGGACAC TGCAGTCTAC TATTGCGCTA
2701 AAGACTATGA AGGTACTGGT TATGCTTTCG ACATATGGGG TCAAGGTACT ATGGTCACCG
2761 TCTCTAGTGC CTCCACCAAG GGCCCATCGG TCTTCCCCCT GGCACCCTCC TCCAAGAGCA
2821 CCTCTGGGGG CACAGCGGCC CTGGGCTGCC TGGTCAAGGA CTACTTCCCC GAACCGGTGA
2881 CGGTGTCGTG GAACTCAGGC GCCCTGACCA GCGGCGTCCA CACCTTCCCG GCTGTCCTAC
2941 AGTCTAGCGG ACTCTACTCC CTCAGCAGCG TAGTGACCGT GCCCTCTTCT AGCTTGGGCA
3001 CCCAGACCTA CATCTGCAAC GTGAATCACA AGCCCAGCAA CACCAAGGTG GACAAGAAAG
3061 TTGAGCCCAA ATCTTGTGCG GCCGCTCATC ACCACCATCA TCACTCTGCT GAACAAAAAC
3121 TCATCTCAGA AGAGGATCTG AATGGTGCCG CAGATATCAA CGATGATCGT ATGGCTGGCG
3181 CCGCTGAAAC TGTTGAAAGT TGTTTAGCAA AACCCCATAC AGAAAATTCA TTTACTAACG
3241 TCTGGAAAGA CGACAAAACT TTAGATCGTT ACGCTAACTA TGAGGGTTGT CTGTGGAATG
3301 CTACAGGCGT TGTAGTTTGT ACTGGTGACG AAACTCAGTG TTACGGTACA TGGGTTCCTA
3361 TTGGGCTTGC TATCCCTGAA AATGAGGGTG GTGGCTCTGA GGGTGGCGGT TCTGAGGGTG
3421 GCGGTTCTGA GGGTGGCGGT ACTAAACCTC CTGAGTACGG TGATACACCT ATTCCGGGCT
3481 ATACTTATAT CAACCCTCTC GACGGCACTT ATCCGCCTGG TACTGAGCAA AACCCCGCTA
3541 ATCCTAATCC TTCTCTTGAG GAGTCTCAGC CTCTTAATAC TTTCATGTTT CAGAATAATA
3601 GGTTCCGAAA TAGGCAGGGG GCATTAACTG TTTATACGGG CACTGTTACT CAAGGCACTG
3661 ACCCCGTTAA AACTTATTAC CAGTACACTC CTGTATCATC AAAAGCCATG TATGACGCTT
3721 ACTGGAACGG TAAATTCAGA GACTGCGCTT TCCATTCTGG CTTTAATGAA GATCCATTCG
3781 TTTGTGAATA TCAAGGCCAA TCGTCTGACC TGCCTCAACC TCCTGTCAAT GCTGGCGGCG
3841 GCTCTGGTGG TGGTTCTGGT GGCGGCTCTG AGGGTGGTGG CTCTGAGGGT GGCGGTTCTG
3901 AGGGTGGCGG CTCTGAGGGA GGCGGTTCCC GTGGTGGCTC TGGTTCCGGT GATTTTGATT
3961 ATGAAAAGAT GGCAAACGCT AATAAGGGGG CTATGACCGA AAATGCCGAT GAAAACGCGC
4021 TACAGTCTGA CGCTAAAGGC AAACTTGATT CTGTCGCTAC TGATTACGGT GCTGCTATCG
4081 ATGGTTTCAT TGGTGACGTT TCCGGCCTTG CTAATGGTAA TGGTGCTACT GGTGATTTTG
4141 CTGGCTCTAA TTCCCAAATG GCTCAAGTCG GTGACGGTGA TAATTCACCT TTAATGAATA
4201 ATTTCCGTCA ATATTTACCT TCCCTCCCTC AATCGGTTGA ATGTCGCCCT TTTGTCTTTA
4261 GCGCTGGTAA ACCATATGAA TTTTCTATTG ATTGTGACAA AATAAACTTA TTCCGTGGTG
4321 TCTTTGCGTT TCTTTTATAT GTTGCCACCT TTATGTATGT ATTTTCTACG TTTGCTAACA
4381 TACTGCGTAA TAAGGAGTCT TAATCATGCC AGTTCTTTTG GGTATTCCGT TATTATTGCG
4441 TTTCCTCGGT TTCCTTCTGG TAACTTTGTT CGGCTATCTG CTTACTTTTC TTAAAAAGGG
4501 CTTCGGTAAG ATAGCTATTG CTATTTCATT GTTTCTTGCT CTTATTATTG GGCTTAACTC
4561 AATTCTTGTG GGTTATCTCT CTGATATTAG CGCTCAATTA CCCTCTGACT TTGTTCAGGG
4621 TGTTCAGTTA ATTCTCCCGT CTAATGCGCT TCCCTGTTTT TATGTTATTC TCTCTGTAAA
4681 GGCTGCTATT TTCATTTTTG ACGTTAAACA AAAAATCGTT TCTTATTTGG ATTGGGATAA
4741 ATAATATGGC TGTTTATTTT GTAACTGGCA AATTAGGCTC TGGAAAGACG CTCGTTAGCG
4801 TTGGTAAGAT TCAGGATAAA ATTGTAGCTG GGTGCAAAAT AGCAACTAAT CTTGATTTAA
4861 GGCTTCAAAA CCTCCCGCAA GTCGGGAGGT TCGCTAAAAC GCCTCGCGTT CTTAGAATAC
4921 CGGATAAGCC TTCTATATCT GATTTGCTTG CTATTGGGCG CGGTAATGAT TCCTACGATG
4981 AAAATAAAAA CGGCTTGCTT GTTCTCGATG AGTGCGGTAC TTGGTTTAAT ACCCGTTCTT
5041 GGAATGATAA GGAAAGACAG CCGATTATTG ATTGGTTTCT ACATGCTCGT AAATTAGGAT
5101 GGGATATTAT TTTTCTTGTT CAGGACTTAT CTATTGTTGA TAAACAGGCG CGTTCTGCAT
5161 TAGCTGAACA TGTTGTTTAT TGTCGTCGTC TGGACAGAAT TACTTTACCT TTTGTCGGTA
5221 CTTTATATTC TCTTATTACT GGCTCGAAAA TGCCTCTGCC TAAATTACAT GTTGGCGTTG
5281 TTAAATATGG CGATTCTCAA TTAAGCCCTA CTGTTGAGCG TTGGCTTTAT ACTGGTAAGA
5341 ATTTGTATAA CGCATATGAT ACTAAACAGG CTTTTTCTAG TAATTATGAT TCCGGTGTTT
5401 ATTCTTATTT AACGCCTTAT TTATCACACG GTCGGTATTT CAAACCATTA AATTTAGGTC
5461 AGAAGATGAA ATTAACTAAA ATATATTTGA AAAAGTTTTC TCGCGTTCTT TGTCTTGCGA
5521 TTGGATTTGC ATCAGCATTT ACATATAGTT ATATAACCCA ACCTAAGCCG GAGGTTAAAA
5581 AGGTAGTCTC TCAGACCTAT GATTTTGATA AATTCACTAT TGACTCTTCT CAGCGTCTTA
5641 ATCTAAGCTA TCGCTATGTT TTCAAGGATT CTAAGGGAAA ATTAATTAAT AGCGACGATT
5701 TACAGAAGCA AGGTTATTCA CTCACATATA TTGATTTATG TACTGTTTCC ATTAAAAAAG
5761 GTAATTCAAA TGAAATTGTT AAATGTAATT AATTTTGTTT TCTTGATGTT TGTTTCATCA
5821 TCTTCTTTTG CTCAGGTAAT TGAAATGAAT AATTCGCCTC TGCGCGATTT TGTAACTTGG
5881 TATTCAAAGC AATCAGGCGA ATCCGTTATT GTTTCTCCCG ATGTAAAAGG TACTGTTACT
5941 GTATATTCAT CTGACGTTAA ACCTGAAAAT CTACGCAATT TCTTTATTTC TGTTTTACGT
6001 GCTAATAATT TTGATATGGT TGGTTCAATT CCTTCCATAA TTCAGAAGTA TAATCCAAAC
6061 AATCAGGATT ATATTGATGA ATTGCCATCA TCTGATAATC AGGAATATGA TGATAATTCC
6121 GCTCCTTCTG GTGGTTTCTT TGTTCCGCAA AATGATAATG TTACTCAAAC TTTTAAAATT
6181 AATAACGTTC GGGCAAAGGA TTTAATACGA GTTGTCGAAT TGTTTGTAAA GTCTAATACT
6241 TCTAAATCCT CAAATGTATT ATCTATTGAC GGCTCTAATC TATTAGTTGT TTCTGCACCT
6301 AAAGATATTT TAGATAACCT TCCTCAATTC CTTTCTACTG TTGATTTGCC AACTGACCAG
6361 ATATTGATTG AGGGTTTGAT ATTTGAGGTT CAGCAAGGTG ATGCTTTAGA TTTTTCATTT
6421 GCTGCTGGCT CTCAGCGTGG CACTGTTGCA GGCGGTGTTA ATACTGACCG CCTCACCTCT
6481 GTTTTATCTT CTGCTGGTGG TTCGTTCGGT ATTTTTAATG GCGATGTTTT AGGGCTATCA
6541 GTTCGCGCAT TAAAGACTAA TAGCCATTCA AAAATATTGT CTGTGCCACG TATTCTTACG
6601 CTTTCAGGTC AGAAGGGTTC TATCTCTGTT GGCCAGAATG TCCCTTTTAT TACTGGTCGT
6661 GTGACTGGTG AATCTGCCAA TGTAAATAAT CCATTTCAGA CGATTGAGCG TCAAAATGTA
6721 GGTATTTCCA TGAGCGTTTT TCCTGTTGCA ATGGCTGGCG GTAATATTGT TCTGGATATT
6781 ACCAGCAAGG CCGATAGTTT GAGTTCTTCT ACTCAGGCAA GTGATGTTAT TACTAATCAA
6841 AGAAGTATTG CTACAACGGT TAATTTGCGT GATGGACAGA CTCTTTTACT CGGTGGCCTC
6901 ACTGATTATA AAAACACTTC TCAAGATTCT GGCGTACCGT TCCTGTCTAA AATCCCTTTA
6961 ATCGGCCTCC TGTTTAGCTC CCGCTCTGAT TCCAACGAGG AAAGCACGTT ATACGTGCTC
7021 GTCAAAGCAA CCATAGTACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTGTGGTGGT
7081 TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT
7141 CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATC GGGGGCTCCC
7201 TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC AAAAAACTTG ATTTGGGTGA
7261 TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT CGCCCTTTGA CGTTGGAGTC
7321 CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA ACACTCAACC CTATCTCGGG
7381 CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGAA CCACCATCAA ACAGGATTTT
7441 CGCCTGCTGG GGCAAACCAG CGTGGACCGC TTGCTGCAAC TCTCTCAGGG CCAGGCGGTG
7501 AAGGGCAATC AGCTGTTtCC CGTCTCACTG GTGAAAAGAA AAACCACCCT GGATCCAAGC
7561 TTGCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA
7621 TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT
7681 GAAAAAGGAA GAGTATGAGT ATTCAACATT TCCGTGTCGC CCTTATTCCC TTTTTTGCGG
7741 CATTTTGCCT TCCTGTTTTT GCTCACCCAG AAACGCTGGT GAAAGTAAAA GATGCTGAAG
7801 ATCAGTTGGG CGCACGAGTG GGTTACATCG AACTGGATCT CAACAGCGGT AAGATCCTTG
7861 AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA TGATGAGCAC TTTTAAAGTT CTGCTATGTC
7921 ATACACTATT ATCCCGTATT GACGCCGGGC AAGAGCAACT CGGTCGCCGG GCGCGGTATT
7981 CTCAGAATGA CTTGGTTGAG TACTCACCAG TCACAGAAAA GCATCTTACG GATGGCATGA
8041 CAGTAAGAGA ATTATGCAGT GCTGCCATAA CCATGAGTGA TAACACTGCG GCCAACTTAC
8101 TTCTGACAAC GATCGGAGGA CCGAAGGAGC TAACCGCTTT TTTGCACAAC ATGGGGGATC
8161 ATGTAACTCG CCTTGATCGT TGGGAACCGG AGCTGAATGA AGCCATACCA AACGACGAGC
8221 GTGACACCAC GATGCCTGTA GCAATGCCAA CAACGTTGCG CAAACTATTA ACTGGCGAAC
8281 TACTTACTCT AGCTTCCCGG CAACAATTAA TAGACTGGAT GGAGGCGGAT AAAGTTGCAG
8341 GACCACTTCT GCGCTCGGCC CTTCCGGCTG GCTGGTTTAT TGCTGATAAA TCTGGAGCCG
8401 GTGAGCGTGG GTCTCGCGGT ATCATTGCAG CACTGGGGCC AGATGGTAAG CCCTCCCGTA
8461 TCGTAGTTAT CTACACGACG GGGAGTCAGG CAACTATGGA TGAACGAAAT AGACAGATCG
8521 CTGAGATAGG TGCCTCACTG ATTAAGCATT GGTAACTGTC AGACCAAGTT TACTCATATA
8581 TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG GATCTAGGTG AAGATCCTTT
8641 TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC GTTCCACTGT ACGTAAGACC
8701 CCCAAGCTTG TCGACTGAAT GGCGAATGGC GCTTTGCCTG GTTTCCGGCA CCAGAAGCGG
8761 TGCCGGAAAG CTGGCTGGAG TGCGATCTTC CTGAGGCCGA TACTGTCGTC GTCCCCTCAA
8821 ACTGGCAGAT GCACGGTTAC GATGCGCCCA TCTACACCAA CGTAACCTAT CCCATTACGG
8881 TCAATCCGCC GTTTGTTCCC ACGGAGAATC CGACGGGTTG TTACTCGCTC ACATTTAATG
8941 TTGATGAAAG CTGGCTACAG GAAGGCCAGA CGCGAATTAT TTTTGATGGC GTTCCTATTG
9001 GTTAAAAAAT GAGCTGATTT AACAAAAATT TAACGCGAAT TTTAACAAAA TATTAACGTT
9061 TACAATTTAA ATATTTGCTT ATACAATCTT CCTGTTTTTG GGGCTTTTCT GATTATCAAC
9121 CGGGGTACAT ATGATTGACA TGCTAGTTTT ACGATTACCG TTCATCGATT CTCTTGTTTG
9181 CTCCAGACTC TCAGGCAATG ACCTGATAGC CTTTGTAGAT CTCTCAAAAA TAGCTACCCT
9241 CTCCGGCATG AATTTATCAG CTAGAACGGT TGAATATCAT ATTGATGGTG ATTTGACTGT
9301 CTCCGGCCTT TCTCACCCTT TTGAATCTTT ACCTACACAT TACTCAGGCA TTGCATTTAA
9361 AATATATGAG GGTTCTAAAA ATTTTTATCC TTGCGTTGAA ATAAAGGCTT CTCCCGCAAA
9421 AGTATTACAG GGTCATAATG TTTTTGGTAC AACCGATTTA GCTTTATGCT CTGAGGCTTT
9481 ATTGCTTAAT TTTGCTAATT CTTTGCCTTG CCTGTATGAT TTATTGGATG TT

[0159]

TABLE 200
Enzymes that either cut 15 or more human GLGs or have 5 + -base recognition in FR3
Typical entry:
REname Recognition #sites
GLGid#:base# GLGid#:base# GLGid#:base#
BstEII Ggtnacc     2
1:  3   48:  3
There are 2 hits at base# 3
MaeIII gtnac       36
1:  4    2:  4    3:  4    4:  4    5:  4    6:  4
7:  4    8:  4    9:  4   10:  4   11:  4   37:  4
37: 58   38:  4   38: 58   39:  4   39: 58   40:  4
40: 58   41:  4   41: 58   42:  4   42: 58   43:  4
43: 58   44:  4   44: 58   45:  4   45: 58   46:  4
46: 58   47:  4   47: 58   48:  4   49:  4   50: 58
There are 24 hits at base# 4
Tsp45I gtsac       33
1:  4    2:  4    3:  4    4:  4    5:  4    6:  4
7:  4    8:  4    9:  4   10:  4   11:  4   37:  4
7: 58   38:  4   38: 58   39: 58   40:  4   40: 58
41: 58   42: 58   43:  4   43: 58   44:  4   44: 58
45:  4   45: 58   46:  4   46: 58   47:  4   47: 58
48:  4   49:  4   50: 58
There are 21 hits at base# 4
1-IphI tcacc       45
1:  5    2:  5    3:  5    4:  5    5:  5    6:  5
7:  5    8:  5   11:  5   12:  5   12: 11   13:  5
14:  5   15:  5   16:  5   17:  5   18:  5   19:  5
20:  5   21:  5   22:  5   23:  5   24:  5   25:  5
26:  5   27:  5   28:  5   29:  5   30:  5   31:  5
32:  5   33:  5   34:  5   35:  5   36:  5   37:  5
38:  5   40:  5   43:  5   44:  5   45:  5   46:  5
47:  5   48:  5   49:  5
There are 44 hits at base# 5
NlaIII CATG        26
1:  9    1: 42    2: 42    3:  9    3: 42    4:  9
4: 42    5:  9    5: 42    6: 42    6: 78    7:  9
7: 42    8: 21    8: 42    9: 42   10: 42   11: 42
12: 57   13: 48   13: 57   14: 57   31: 72   38:  9
48: 78   49: 78
There are 11 hits at base# 42
There are 1 hits at base# 48 Could cause raggedness.
BsaJI Ccnngg       37
1: 14    2: 14    5: 14    6: 14    7: 14    8: 14
8: 65    9: 14   10: 14   11: 14   12: 14   13: 14
14: 14   15: 65   17: 14   17: 65   18: 65   19: 65
20: 65   21: 65   22: 65   26: 65   29: 65   30: 65
33: 65   34: 65   35: 65   37: 65   38: 65   39: 65
40: 65   42: 65   43: 65   48: 65   49: 65   50: 65
51: 14
There are 23 hits at base# 65
There are 14 hits at base# 14
AluI AGct          42
1: 47    2: 47    3: 47    4: 47    5: 47    6: 47
7: 47    8: 47    9: 47   10: 47   11: 47   16: 63
23: 63   24: 63   25: 63   31: 63   32: 63   36: 63
37: 47   37: 52                     38: 47   38: 52   39: 47   39: 52
40: 47   40: 52                     41: 47   41: 52   42: 47   42: 52
43: 47   43: 52                     44: 47   44: 52   45: 47   45: 52
46: 47   46: 52                     47: 47   47: 52   49: 15   50: 47
There are 23 hits at base# 47
There are 11 hits at base# 52                   Only 5 bases from 47
BlpI GCtnagc       21
1: 48    2: 48    3: 48    5:48    6:48    7:48
8: 48    9: 48   10: 48   11:48   37:48   38:48
39: 48   40: 48   41: 48   42:48   43:48   44:48
45: 48   46: 48   47: 48
There are 21 hits at base# 48
MwoI GCNNNNNnngc   19
1: 48    2: 28   19: 36   22: 36   23: 36   24: 36
25: 36   26: 36   35: 36   37: 67   39: 67   40: 67
41: 67   42: 67   43: 67   44: 67   45: 67   46: 67
47: 67
There are 10 hits at base# 67
There are 7 hits at base# 36
DdeI Ctnag         71
1: 49    1: 58    2: 49    2: 58    3: 49    3: 58
3: 65    4: 49    4: 58    5: 49    5: 58    5: 65
6: 49    6: 58    6: 65    7: 49    7: 58    7: 65
8: 49    8: 58    9: 49    9: 58    9: 65   10: 49
10: 58   10: 65                     11: 49   11: 58   11: 65   15: 58
16: 58   16: 65                     17: 58   18: 58   20: 58   21: 58
22: 58   23: 58   23: 65   24: 58   24: 65   25: 58
25: 65                     26: 58   27: 58   27: 65   28: 58   30: 58
31: 58   31: 65                     32: 58   32: 65   35: 58   36: 58
36: 65                     37: 49   38: 49   39: 26   39: 49   40: 49
41: 49   42: 26   42: 49   43: 49   44: 49   45: 49
46: 49   47: 49   48: 12   49: 12   51: 65
There are 29 hits at base# 58
There are 22 hits at base# 49                   Only nine base from 58
There are 16 hits at base# 65                   Only seven bases from 58
BglII Agatct       11
1: 61    2: 61    3: 61    4: 61    5: 61    6: 61
7: 61    9: 61   10: 61   11: 61   51: 47
There are 10 hits at base# 61
BstYI Rgatcy       12
1: 61    2: 61    3: 61    4: 61    5: 61    6: 61
7: 61    8: 61    9: 61   10: 61   11: 61   51: 47
There are 11 hits at base# 61
Hpy188I TCNga      17
1: 64    2: 64    3: 64    4: 64    5: 64    6: 64
7: 64    8: 64    9: 64   10: 64   11: 64   16: 57
20: 57   27: 57   35: 57   48: 67   49: 67
There are 11 hits at base# 64
There are 4 hits at base# 57
There are 2 hits at base# 67 Could be ragged                  .
MslI CAYNNnnRTG    44
1: 72    2: 72    3: 72    4: 72    5: 72    6: 72
7: 72    8: 72    9: 72   10: 72   11: 72   15: 72
17: 72   18: 72   19: 72   21: 72   23: 72   24: 72
25: 72   26: 72   28: 72   29: 72   30: 72   31: 72
32: 72   33: 72   34: 72   35: 72   36: 72   37: 72
39: 72   39: 72   40: 72   41: 72   42: 72   43: 72
44: 72   45: 72   46: 72   47: 72   48: 72   49: 72
50: 72   51: 72
There are 44 hits at base# 72
BsiEI CGRYcg       23
1: 74    3: 74    4: 74    5: 74    7: 74    8: 74
9: 74   10: 74   11: 74   17: 74   22: 74   30: 74
33: 74   34: 74   37: 74   38: 74   39: 74   40: 74
41: 74   42: 74   45: 74   46: 74   47: 74
There are 23 hits at base# 74
EaeI Yggccr        23
1: 74    3: 74    4: 74    5: 74    7: 74    8: 74
9: 74   10: 74   11: 74   17: 74   22: 74   30: 74
33: 74   34: 74   37: 74   38: 74   39: 74   40: 74
41: 74   42: 74   45: 74   46: 74   47: 74
There are 23 hits at basef 74
EagI Cggccg        23
1: 74    3: 74    4: 74    5: 74    7: 74    8: 74
9: 74   10: 74   11: 74   17: 74   22: 74   30: 74
33: 74   34: 74   37: 74   38: 74   39: 74   40: 74
41: 74   42: 74   45: 74   46: 74   47: 74
There are 23 hits at base# 74
HaeIII GGcc        27
1: 75    3: 75    4: 75    5: 75    7: 75    8: 75
9: 75   10: 75   11: 75   16: 75   17: 75   20: 75
22: 75   30: 75   33: 75   34: 75   37: 75   38: 75
39: 75   40: 75   41: 75   42: 75   45: 75   46: 75
47: 75   48: 63   49: 63
There are 25 hits at base# 75
Bst4CI ACNgt 65° C.                                                        63 Sites There is a third isoschismer
1: 86    2: 86    3: 86    4: 86    5: 86    6: 86
7: 34    7: 86    8: 86    9: 86   10: 86   11: 86
12: 86   13: 86   14: 96   15: 36   15: 86   16: 53
16: 86   17: 36   17: 86   18: 86   19: 86   20: 53
20: 86   21: 36   21: 86   22:  0   22: 66   23: 86
24: 86   25: 86   26: 86   27: 53   27: 86   28: 36
28: 86   29: 86   30: 86   31: 86   32: 86   33: 36
33: 86   34: 86   35: 53   35: 86   36: 86   37: 86
38: 86   39: 86   40: 86   41: 86   42: 86   43: 86
44: 86   45: 86   46: 86   47: 86   48: 86   49: 86
50: 86   51:  0   51: 86
There are 51 hits at base# 86                   All the other sites are well away
HpyCH4III ACNgt    63
1: 86    2: 86    3: 86    4: 86    5: 86    6: 86
7: 34    7: 86    8: 86    9: 86   10: 86   11: 86
12: 86   13: 86   14: 86   15: 36   15: 86   16: 53
16: 86   17: 36   17: 86   18: 86   19: 86   20: 53
20: 86   21: 36   21: 86   22:  0   22: 86   23: 86
24: 86   25: 86   26: 86   27: 53   27: 86   28: 36
28: 86   29: 86   30: 86   31: 86   32: 86   33: 36
33: 86   34: 86   35: 53   35: 86   36: 86   37: 86
38: 86   39: 86   40: 86   41: 86   42: 86   43: 86
44: 96   45: 86   46: 86   47: 86   48: 86   49: 86
50: 66   51:  0   51: 96
There are 51 hits at base# 86
Hinf I Gantc       43
2:  2    3:  2    4:  2    5:  2    6:  2    7: 2
8:  2    9:  2    9: 22   10:  2   11:  2   15: 2
16:  2   17:  2   19:  2   19:  2   19: 22   20: 2
21:  2   23:  2   24:  2   25:  2   26:  2   27: 2
28:  2   29:  2   30:  2   31:  2   32:  2   33: 2
33: 22   34: 22   35:  2   36:  2   37:  2   38: 2
40:  2   43:  2   44:  2   45:  2   46:  2   47: 2
50: 60
There are 38 hits at base# 2
MlyI GAGTCNNNNNn   18
2:  2    3:  2    4:  2    5:  2    6:  2    7:  2
8:  2    9:  2   10:  2   11:  2   37:  2   38:  2
40:  2   43:  2   44:  2   45:  2   46:  2   47:  2
There are 18 hits at base# 2
PleI gagtc         18
2:  2    3:  2    4:  2    5:  2    6:  2    7:  2
8:  2    9:  2   10:  2   11:  2   37:  2   38:  2
40:  2   43:  2   44:  2   45:  2   46:  2   47:  2
There are 18 hits at base# 2
AciI Ccgc          24
2: 26    9: 14   10: 14   11: 14   27: 74   37: 62
37: 65   38: 62   39: 65   40: 62   40: 65   41: 65
42: 65   43: 62   43: 65   44: 62   44: 65   45: 62
46: 62   47: 62   47: 65   48: 35   48: 74   49: 74
There are 8 hits at base# 62
There are 8 hits at base# 65
There are 3 hits at base# 14
There are 3 hits at base# 74
There are 1 hits at base# 26
There are 1 hits at base# 35
-″- Gcgg           11
8: 91    9: 16   10: 16   11: 16   37: 67   39: 67
40: 67   42: 67   43: 67   45: 67   46: 67
There are 7 hits at base# 67
There are 3 hits at base#16
There are 1 hits at base#91
BsiIHKAI GWGCWc    20
2: 30    4: 30    6: 30    7: 30    9: 30   10: 30
12: 89   13: 89   14: 89   37: 51   38: 51   39: 51
40: 51   41: 51   42: 51   43: 51   44: 51   45: 51
46: 51   47: 51
There are 11 hits at base# 51
Bsp1286I GDGCHc    20
2: 30    4: 30    6: 30    7: 30    9: 30   10: 30
12: 89   13: 89   14: 89   37: 51   38: 51   39: 51
40: 51   41: 51   42: 51   43: 51   44: 51   45: 51
46: 51   47: 51
There are 11 hits at base# 51
HgiAI GWGCWc       20
2: 30    4: 30    6: 30    7: 30    9: 30   10: 30
12: 89   13: 89   14: 89   37: 51   38: 51   39: 51
40: 51   41: 51   42: 51   43: 51   44: 51   45: 51
46: 51   47: 51
There are 11 hits at base# 51
BsoFI GCngc        26
2: 53    3: 53    5: 53    6: 53    7: 53    8: 53
8: 91    9: 53   10: 53   11: 53   31: 53   36: 36
37: 64   39: 64   40: 64   41: 64   42: 64   43: 64
44: 64   45: 64   46: 64   47: 64   48: 53   49: 53
50: 45   51: 53
There are 13 hits at base# 53
There are 10 hits at base# 64
TseI Gcwgc         17
2: 53    3: 53    5: 53    6: 53    7: 53    8: 53
9: 53   10: 53   11: 53   31: 53   36: 36   45: 64
46: 64   48: 53   49: 53   50: 45   51: 53
There are 13 hits at base# 53
MnlI gagg          34
3: 67    3: 95    4: 51    5: 16    5: 67    6: 67
7: 67    8: 67    9: 67   10: 67   11: 67   15: 67
16: 67   17: 67   19: 67   20: 67   21: 67   22: 67
23: 67   24: 67   25: 67   26: 67   27: 67   28: 67
29: 67   30: 67   31: 67   32: 67   33: 67   34: 67
35: 67   36: 67   50: 67   51: 67
There are 31 hits at base#67
HpyCH4V TGCa       34
5: 90    6: 90   11: 90   12: 90   13: 90   14: 90
15: 44   16: 44   16: 90   17: 44   16: 90   19: 44
20: 44   21: 44   22: 44   23: 44   24: 44   25: 44
26: 44   27: 44   27: 90   28: 44   29: 44   33: 44
34: 44   35: 44   35: 90   36: 39   48: 44   49: 44
50: 44   50: 90   51: 44   51: 52
There are 21 hits at base# 44
There are 1 hits at base# 52
AccI GTmkac        13 5-base recognition
7: 37   11: 24   37: 16   38: 16   39: 16   40: 16
41: 16   42: 16   43: 16   44: 16   45: 16   46: 16
47: 16
There are 11 hits at base# 16
SacII CCGCgg       8 6-base recognition
9: 14   10: 14   11: 14   37: 65   39: 65   40: 65
42: 65   43: 65
There are 5 hits at base# 65
There are 3 hits at base# 14
TfiI Gawtc         24
9: 22   15:  2   16:  2   17:  2   18:  2   19:  2
19: 22   20:  2   21:  2   23:  2   24:  2   25:  2
26:  2   27:  2   28:  2   29:  2   30:  2   31:  2
32:  2   33:  2   33: 22   34: 22   35:  2   36:  2
There are 20 hits at base# 2
BsmAI Nnnnnngagac  19
15: 12   16: 11   20: 11   21: 11   22: 11   23: 11
24: 11   25: 11   26: 11   27: 11   28: 12   28: 56
30: 11   31: 11   32: 11   35: 11   36: 11   44: 87
48: 87
There are 16 hits at base# 11
BpmI ctccag        19
15: 12   16: 12   17: 12   18: 12   20: 12   21: 12
22: 12   23: 12   24: 12   25: 12   26: 12   27: 12
28: 12   30: 12   31: 12   32: 12   34: 12   35: 12
36: 12
There are 19 hits at base# 12
XmnI GAANNnnttc    12
37: 30   38: 30   39: 30   40: 30   41: 30   42: 30
43: 30   44: 30   45: 30   46: 30   47: 30   50: 30
There are 12 hits at base# 30
BsrI NCcagt        12
37: 32   38: 32   39: 32   40: 32   41: 32   42: 32
43: 32   44: 32   45: 32   46: 32   47: 32   50: 32
There are 12 hits at base# 32
BanII GRGCYc       11
37: 51   38: 51   39: 51   40: 51   41: 51   42: 51
43: 51   44: 51   45: 51   46: 51   47: 51
There are 11 hits at base# 51
Ecl136I GAGctc     11
37: 51   38: 51   39: 51   40: 51   41: 51   42: 51
43: 51   44: 51   45: 51   46: 51   47: 51
There are 11 hits at base# 51
SacI GAGCTc        11
37: 51   38: 51   39: 51   40: 51   41: 51   42: 51
43: 51   44: 51   45: 51   46: 51   47: 51
There are 11 hits at base# 51

[0160]

TABLE 206
! Synthetic 3-23 FR3 of human heavy chains showning positions of possible cleavage sites
! Sites engineered into the synthetic gene are shown in upper case DNA
! with the RE name between vertical bars (as in Xibal I).
! RERSs frequently found in GLGs are shown below the synthetic sequence
! with the name to the right (as in gtn ac=MaeIII(24), indicating that
! 24 of the 51 GLGs contain the site)
!                                                              |---FR3---
!                                                                89  90 (codon # in
!                                                                 R   F  synthetic 3-23)
|cgc|ttc|  6
! Allowed DNA                                                        |cgn|tty|
!                                                                    |agr|
!                                                                      ga ntc = HinfI (38)
!                                                                      ga gtc = PleI (18)
!                                                                      ga wtc = TfiI (20)
!                                                                         gtn ac = MaeIII (24)
!                                                                         gts ac = Tsp45I (21)
!                                                                          tc acc = HphI (44)
!       --------FR3-------------------------------------------------
!          91  92  93  94  95  96  97  98  99  100  101  102  103  104  105
!          T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M
|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|  51
!allowed|acn|ath|tcn|cgn|gay|aay|tcn|aar|aay|acn|ttr|tay|ttr|car|atg|
!              |agy|agr|       |agy|            |ctn|   |ctn|
!              |     ga|gac = BsmAI (16)                      ag ct = AluI (23)
!             c|tcc ag = BpmI (19)                                g ctn agc = BlpI (21)
!              |       |               g aan nnn ttc = XmnI (12)
!              |XbaI  |                                  tg ca = HpyCH4V (21)
!
!         ---FR3----------------------------------------------------->|
!          106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
!           N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   K
|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa|  96
!allowed|aay|tcn|ttr|cgn|gcn|gar|gay|acn|gcn|gtn|tay|tay|tgy|gcn|aar|
!           |agy|ctn|agr|             |       |
!               |      |   cc nng g = BsaJI (23)        ac ngt = Bst4cI (51)
!               |     aga tct = BglII (10)     |        ac ngt = HpyCH4III (51)
!               |     Rga tcY = BstYI (11)     |        ac ngt = TaaI (51)
!               |      |            c ayn nnn rtc = MslI (44)
!               |      |               cg ryc g = BsiEI (23)
!               |      |               yg gcc r = EaeI (23)
!               |      |               cg gcc g = EagI (23)
!               |      |               |g gcc = HaeIII (25)
!               |      |      gag g = MnlI (31)|
!               |AflII |               |PstI |

[0161]

TABLE 217
Human HG GLG FR1 Sequences
VH Exon-Nucleotide sequence alignment
VH1
1-02   CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG
       GTC TCC TGC AAG GCT TCT GGA TAC ACC TTC ACC
1-03   cag gtC cag ctT gtg cag tct ggg gct gag gtg aag aag cct ggg gcc tca gtg aag
       gtT tcc tgc aag gct tct gga tac acc ttc acT
1-08   cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg gcc tca gtg aag
       gtc tcc tgc aag gct tct gga tac acc ttc acc
1-18   cag gtT cag ctg gtg cag tct ggA gct gag gtg aag cag cct ggg gcc tca gtg aag
       gtc tcc tgc aag gct tct ggT tac acc ttT acc
1-24   cag gtC cag ctg gtA cag tct ggg gct gag gtg aag aag cct ggg gcc tca gtg aag
       gtc tcc tgc aag gTt tcC gga tac acc Ctc acT
1-45   cag Atg cag ctg gtg cag tct ggg gct gag gtg aag aag Act ggg Tcc tca gtg aag
       gtT tcc tgc aag gct tcC gga tac acc ttc acc
1-46   cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg gcc tca gtg aag
       gtT tcc tgc aag gcA tct gga tac acc ttc acc
1-58   caA Atg cag ctg gtg cag tct ggg cct gag gtg aag aag cct ggg Acc tca gtg aag
       gtc tcc tgc aag gct tct gga tTc acc ttT acT
1-69   cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg Tcc tcG gtg aag
       gtc tcc tgc aag gct tct gga GGc acc ttc aGc
1-e    cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg Tcc tcG gtg aag
       gtc tcc tgc aag gct tct gga GGc acc ttc aGc
1-f    Gag gtC cag ctg gtA cag tct ggg gct gag gtg aag aag cct ggg gcT Aca gtg aaA
       Atc tcc tgc aag gTt tct gga tac acc ttc acc
VH2
2-05   CAG ATC ACC TTG AAG GAG TCT GGT CCT ACG CTG GTG AAA CCC ACA CAG ACC CTC ACG
       CTG ACC TGC ACC TTC TCT GGG TTC TCA CTC AGC
2-26   cag Gtc acc ttg aag gag tct ggt cct GTg ctg gtg aaa ccc aca Gag acc ctc acg
       ctg acc tgc acc Gtc tct ggg ttc tca ctc agc
2-70   cag Gtc acc ttg aag gag tct ggt cct Gcg ctg gtg aaa ccc aca cag acc ctc acA
       ctg acc tgc acc ttc tct ggg ttc tca ctc agc
VH3
3-07   GAG GTG CAG CTC GTG GAG TCT GGG GGA GGC TTG GTC CAG CCT GGG GGG TCC CTG AGA
       CTC TCC TGT GCA GCC TCT GGA TTC ACC TTT AGT
3-09   gaA gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggC Agg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttt GAt
3-11   Cag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc Aag cct ggA ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttC agt
3-13   gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttC agt
3-15   gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA Aag cct ggg ggg tcc ctT aga
       ctc tcc tgt gca gcc tct gga ttc acT ttC agt
3-20   gag gtg cag ctg gtg gag tct ggg gga ggT Gtg gtA cGg cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttt GAt
3-21   gag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc Aag cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttC agt
3-23   gag gtg cag ctg Ttg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tac ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttt agC
3-30   Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttc agt
3-30.3 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttC acc ttG agt
3-30.5 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttC agt
3-33   Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga
       ctc tcc tgt gca gcG tct gga ttc acc ttC agt
3-43   gaA gtg cag ctg gtg gag tct ggg gga gTc Gtg gtA cag cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttt GAt
3-48   gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttc agt
3-49   gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag ccA ggg Cgg tcc ctg aga
       ctc tcc tgt Aca gcT tct qga ttc acc ttt Ggt
3-53   gag gtg cag ctg gtg gag Act ggA gga ggc ttg Atc cag cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct ggG ttc acc GtG agt
3-64   gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttG agt
3-66   gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc GtC agt
3-72   gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggA ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttC agt
3-73   gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tcc ctg aAa
       ctc tcc tgt gca gcc tct ggG ttc acc ttc agt
3-74   gag gtg cag ctg gtg gag tcC ggg gga ggc ttA gtT cag cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc ttG agt
3-d    gag gtg cag ctg gtg gag tct Cgg gga gTc ttg gtA cag cct ggg ggg tcc ctg aga
       ctc tcc tgt gca gcc tct gga ttc acc GtC agt
VH4
4-04   GAG GTG GAG CTG GAG GAG TGG CCC GGA CGA CTG GTG AAG CCT TCG GGG ACC CTG TCC
       CTC AGC TGC GGT GTC TCT GGT CCC TCC ATC AGC
4-28   cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAG acc ctg tcc
       ctc acc tgc gct gtc tct ggt TAc tcc atc agc
4-30.1 cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA GAg acc ctg tcc
       ctc acc tgc Act gtc tct ggt ggc tcc atc agc
4-30.2 cag Ctg cag ctg cag gag tcG ggc Tca gga ctg gtg aag cct tcA GAg acc ctg tcc
       ctc acc tgc gct gtc tct ggt ggc tcc atc agc
4-30.4 cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA GAg acc ctg tcc
       ctc acc tgc Act gtc tct ggt ggc tcc atc agc
4-31   cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tcc
       ctc acc tgc Act gtc tct ggt ggc tcc atc agc
4-34   cag gtg cag ctA cag Cag tGg ggc Gca gga ctg Ttg aag cct tcg gAg acc ctg tcc
       ctc acc tgc gct gtc tAt ggt ggG tcc Ttc agT
4-39   cag Ctg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc
       ctc acc tgc Act gtc tct ggt ggc tcc atc agc
4-59   cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc
       ctc acc tgc Act gtc tct ggt ggc tcc atc agT
4-61   cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc
       ctc acc tgc Act gtc tct ggt ggc tcc Gtc agc
4-b    cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc
       ctc acc tgc gct gtc tct ggt TAc tcc atc agc
VH5
5-51   GAG GTG CAG CTG GTG CAG TCT GGA GCA GAG GTG AAA AAG CCC GGG GAG TCT CTG AAG
       ATC TCC TGT AAG GGT TCT GGA TAC AGC TTT ACC
5-a    gaA gtg cag ctg gtg cag tct gga gca gag gtg aaa aag ccc ggg gag tct ctg aGg
       atc tcc tgt aag ggt tct gga tac agc ttt acc
VH6
6-1    CAG GTA CAG CTG CAG CAG TCA GCT CCA GGA CTG GTG AAG CCC TCG CAG ACC CTC TCA
       CTC ACC TGT GCC ATC TCC GGG GAC AGT GTC TCT
VH7
7-4.1  CAG GTG CAG CTG GTG CAA TCT GGG TCT GAG TTG AAG AAG CCT GGG GCC TCA GTG AAG
       GTT TCC TGC AAG GCT TCT GGA TAC ACC TTC ACT

[0162]

TABLE 220
RERS Sites in Human HC GLG FR1s where there are
at least 20 GLGs cut
Bsg1 GTGCAG                      71 (cuts 16 14 bases to right)
1:  4     1: 13     2: 13     3:  4     3:13     4: 13
6: 13     7:  4     7: 13     8: 13     9:  4     9: 13
10:  4   10: 13   15:  4   15: 65   16:  4   16: 65
27:  4   17: 65   18:  4   18: 65   19:  4   19: 65
20:  4   20: 65   21:  4   21: 65   22:  4   22: 65
23:  4   23: 65   24:  4   24: 65   25:  4   25: 65
26:  4   26: 65   27:  4   27: 65   28:  4   28: 65
29:  4   30:  4   30: 65   31:  4   31: 65   32:  4
32: 65   33:  4   33: 65   34:  4   34: 65   35:  4
35: 65   36:  4   36: 65   37:  4   38:  4   39:  4
41:  4   42:  4   43:  4   45:  4   46:  4   47:  4
48:  4   48: 13   49:  4   49: 13   51:  4
There are 39 hits at base# 4
There are 21 hits at base#65
-″- ctgcac                     9
12: 63   13: 63   14: 63   39: 63   41: 63   42: 63
44: 63   45: 63   46: 63
BbvI GCAGC                    65
1:  6     3:  6     6:  6     7:  6     8:  6     9:  6
10:  6   15:  6   15: 67   16:  6   16: 67   17:  6
17: 67   18:  6   18: 67   19:  6   19: 67   20:  6
20: 67   21:  6   21: 67   22:  6   22: 67   23:  6
23: 67   24:  6   24: 67   25:  6   25: 67   26:  6
26: 67   27:  6   27: 67   28:  6   28: 67   29:  6
30:  6   30: 67   31:  6   31: 67   32:  6   32: 67
33:  6   33: 67   34:  6   34: 67   35:  6   35: 67
36:  6   36: 67   37:  6   38:  6   39:  6   40:  6
41:  6   42:  6   43:  6   44:  6   45:  6   46:  6
47:  6   48:  6   49:  6   50: 12   51:  6
There are 43 hits at base#6 Bolded sites very near sites
listed below
There are 21 hits at base#67
-″- gctgc                    13
37:  9   38:  9   39:  9   40:  3   40:  9   41:  9
42:  9   44:  3   44:  9   45:  9   46:  9   47:  9
50:  9
There are 11 hits at base#9
BsoFI GCngc 78
1:  6    3:  6    6:  6    7:  6  8:  6    9:  6
10:  6   15:  6   15: 67   16:  6   16: 67   17:  6
17: 67   18:  6   18: 67   19:  6   19: 67   20:  6
20: 67   21:  6   21: 67   22:  6   22: 67   23:  6
23: 67   24:  6   24: 67   25:  6   25: 67   26:  6
26: 67   27:  6   27: 67   28:  6   28: 67   29:  6
30:  6   30: 67   31:  6   31: 67   32:  6   32: 67
33:  6   33: 67   34:  6   34: 67   35:  6   35: 67
36:  6   36: 67   37:  6   37:  9  38:  6   38:  9
39:  6   39:  9   40:  3   40:  6   40:  9  41:  6
41:  9   42:  6   42:  9   43:  6   44:  3   44:  6
44:  9                    45:  6   45:  9  46:  6   46:  9  47:  6
47:  9                    48:  6   49:  6   50:  9   50: 12   51:  6
There are 43 hits at base#6 These of ten occur together.
There are 11 hits at base#9
There are 2 hits at base#3
There are 21 hits at base#67
TseI Gcwgc                    78
1:  6    3:  6    6:  6    7:  6    8:  6    9:  6
10:  6   15:  6   15: 67   16:  6   16: 67   17:  6
17: 67   18:  6   18: 67   19:  6   19: 67   20:  6
20: 67   21:  6   21: 67   22:  6   22: 67   23:  6
23: 67   24:  6   24: 67   25:  6   25: 67   26:  6
26: 67   27:  6   27: 67   28:  6   28: 67   29:  6
30:  6   30: 67   31:  6   31: 67   32:  6   32: 67
33:  6   33: 67   34:  6   34: 67   35:  6   35: 67
36:  6   36: 67   37:  6   37:  9  38:  6   38:  9
39:  6   39:  9                    40:  3   40:  6   40:  90   41:  6
41:  9                    42:  6   42:  9  43:  6   44:  3   44:  6
44:  9                    45:  6   45:  9  46:  6   46:  9  47:  6
47:  9                    48:  6   49:  6   50:  9   50: 12  51:  6
There are 43 hits at base#6 Often together.
There are 11 hits at base#9
There are 2 hits at base#3
There are 1 hits at base#12
There are 21 hits at base#67
MspAlI CMGckg                    48
1:  7    3:  7    4:  7    5:  7    6:  7    7:  7
8:  7    9:  7   10:  7   11:  7   15:  7   16:  7
17:  7   18:  7   19:  7   20:  7   21:  7   22:  7
23:  7   24:  7   25:  7   26:  7   27:  7   28:  7
29:  7   30:  7   31:  7   32:  7   33:  7   34:  7
35:  7   36:  7   37:  7   38:  7   39:  7   40:  1
40:  7                    41:  7   42:  7   44:  1   44:  7  45:  7
46:  7   47:  7   48:  7   49:  7   50:  7   51:  7
There are 46 hits at base#7
PvuII CAGctg                     48
1:  7    3:  7    4:  7    5:  7    6:  7   7:  7
8:  7    9:  7   10:  7   11:  7   15:  7   16:  7
17:  7   18:  7   19:  7   20:  7   21:  7   22:  7
23:  7   24:  7   25:  7   26:  7   27:  7   28:  7
29:  7   30:  7   31:  7   32:  7   33:  7   34:  7
35:  7   36:  7   37:  7   38:  7   39:  7   40:  1
40:  7                    41:  7   42:  7   44:  1   44:  7  45:  7
46:  7   47:  7   48:  7   49:  7   50:  7   51:  7
There are 46 hits at base#7
There are 2 hits at base#1
AluI AGct                    54
1:  8    2:  8    3:  8    4:  8    4: 24    5:  8
6:  8    7:  8    8:  8    9:  8   10:  8 11:  8
15:  8   16:  8   17:  8   18:  8   19:  8   20:  8
21:  8   22:  8   23:  8   24:  8   25:  8   26:  8
27:  8   28:  8   29:  8   29: 69   30:  8   31:  8
32:  8   33:  8   34:  8   35:  8   36:  8   37:  8
38:  8   39:  8   40:  2   40:  8  41:  8   42:  8
43:  8   44:  2   44:  8  45:  8   46:  8   47:  8
48:  8   48: 82   49:  8   49: 82   50:  8   51:  8
There are 48 hits at base#8
There are 2 hits at base#2
DdeI Ctnag                    48
1: 26    1: 48    2: 26    2: 48    3: 26    3: 48
4: 26    4: 48    5: 26    5: 48    6: 26    6: 48
7: 26    7: 48    8: 26    8: 48    9: 26   10: 26
11: 26   12: 85   13: 85   14: 85   15: 52   16: 52
17: 52   18: 52   19: 52   20: 52   21: 52   22: 52
23: 52   24: 52   25: 52   26: 52   27: 52   28: 52
29: 52   30: 52   31: 52   32: 52   33: 52   35: 30
35: 52   36: 52   40: 24   49: 52   51: 26   51: 48
There are 22 hits at base#52 52 and 48 never together.
There are 9 hits at base#48
There are 12 hits at base#26 26 and 24 never together.
HphI tcacc                    42
1: 86    3: 86    6: 86    7: 86    8: 80 11: 86
12:  5   13:  5   14:  5   15: 80   16: 80   17: 80
18: 80   20: 80   21: 80   22: 80   23: 80   24: 80
25: 80   26: 80   27: 80   28: 80   29: 80   30: 80
31: 80   32: 80   33: 80   34: 80   35: 80   36: 80
37: 59   38: 59   39: 59   40: 59   41: 59   42: 59
43: 59   44: 59   45: 59   46: 59   47: 59   50: 59
There are 22 hits at base#80 80 and 86 never together
There are 5 hits at base#86
There are 12 hits at base#59
BssKI Nccngg                    50
1: 39    2: 39    3: 39    4: 39    5: 39    7: 39
8: 39    9: 39   10: 39   11: 39   15: 39   16: 39
17: 39   18: 39   19: 39   20: 39   21: 29   21: 39
22: 39   23: 39   24: 39   25: 39   26: 39   27: 39
28: 39   29: 39   30: 39   31: 39   32: 39   33: 39
34: 39   35: 19   35: 39   36: 39   37: 24   38: 24
39: 24   41: 24   42: 24   44: 24   45: 24   46: 24
47: 24   48: 39   48: 40  49: 39   49: 40  50: 24
50: 73   51: 39
There are 35 hits at base#39 39 and 40 together twice.
There are 2 hits at base#40
BsaJI Ccnngg                    47
1: 40    2: 40    3: 40    4: 40    5: 40    7: 40
8: 40    9: 40    9: 47   10: 40   10: 47   11: 40
15: 40   18: 40   19: 40   20: 40   21: 40   22: 40
23: 40   24: 40   25: 40   26: 40   27: 40   28: 40
29: 40   30: 40   31: 40   32: 40   34: 40   35: 20
35: 40   36: 40   37: 24   38: 24   39: 24   41: 24
42: 24   44: 24   45: 24   46: 24   47: 24   48: 40
48: 41                    49: 40   49: 41  50: 74   51: 40
There are 32 hits at base#40 40 and 41 together twice
There are 2 hits at base#41
There are 9 hits at base#24
There are 2 hits at base#47
BstNI Ccwgg                    44
PspGI ccwgg
ScrFI ($M.HpaII) CCwgg
1: 40    2: 40    3: 40    4: 40    5: 40    7: 40
8: 40    9: 40   10: 40   11: 40   15: 40   16: 40
17: 40   18: 40   19: 40   20: 40   21: 30   21: 40
22: 40   23: 40   24: 40   25: 40   26: 40   27: 40
28: 40   29: 40   30: 40   31: 40   32: 40   33: 40
34: 40   35: 40   36: 40   37: 25   38: 25   39: 25
41: 25   42: 25   44: 25   45: 25   46: 25   47: 25
50: 25   51: 40
There are 33 hits at base#40
ScrFI CCngg                    50
1: 40    2: 40    3: 40    4: 40    5: 40    7: 40
8: 40    9: 40   10: 40   11: 40   15: 40   16: 40
17: 40   18: 40   19: 40   20: 40   21: 30   21: 40
22: 40   23: 40   24: 40   25: 40   26: 40   27: 40
28: 40   29: 40   30: 40   31: 40   32: 40   33: 40
34: 40   35: 20   35: 40   36: 40   37: 25   38: 25
39: 25   41: 25   42: 25   44: 25   45: 25   46: 25
47: 25   48: 40   48: 41   49: 40   49: 41   50: 25
50: 74   51: 40
There are 35 hits at base#40
There are 2 hits at base#41
Eco01091 RGgnccy                       34
1: 43    2: 43    3: 43    4: 43    5: 43    6: 43
7: 43    8: 43    9: 43   10: 43   15: 46   16: 46
17: 46   18: 46   19: 46   20: 46   21: 46   22: 46
23: 46   24: 46   25: 46   26: 46   27: 46   28: 46
30: 46   31: 46   32: 46   33: 46   34: 46   35: 46
36: 46   37: 46   43: 79   51: 43
There are 22 hits at base#46 46 and 43 never together
There are 11 hits at base#43
NlaIV GGNncc                       71
1: 43    2: 43    3: 43    4: 43    5: 43    6: 43
7: 43    8: 43    9: 43    9: 79   10: 43   10: 79
15: 46   15: 47                    16: 47   17: 46   17: 47  18: 46
18: 47                    19: 46   19: 47  20: 46   20: 47  21: 46
21: 47                    22: 46   22: 47  23: 47   24: 47   25: 47
26: 47   27: 46   27: 47  28: 46   28: 47  29: 47
30: 46   30: 47                    31: 46   31: 47  32: 46   32: 47
33: 46   33: 47                    34: 46   34: 47  35: 46   35: 47
36: 46   36: 47                    37: 21   37: 46   37: 47  37: 79
38: 21   39: 21   39: 79   40: 79   41: 21   41: 79
42: 21   42: 79   43: 79   44: 21   44: 79   45: 21
45: 79   46: 21   46: 79   47: 21   51: 43
There are 23 hits at base#47 46 & 47 often together
There are 17 hits at base#46 There are 11 hits at base#43
Sau96I Ggncc                    70
1: 44    2: 3     2: 44    3: 44    4: 44    5: 3    5: 44    6: 44
7: 44    8: 22    8: 44    9: 44  10: 44  11: 3   12: 22  13: 22
14: 22   15: 33   15: 47   16: 47   17: 47   18: 47   19: 47   20: 47
21: 47   22: 47   23: 33   23: 47   24: 33   24: 47   25: 33   25: 47
26: 33   26: 47   27: 47   28: 47   29: 47   30: 47   31: 33   31: 47
32: 33   32: 47   33: 33   33: 47   34: 33   34: 47   35: 47   36: 47
37: 21   37: 22                    37: 47   38: 21   38: 22  39: 21   39: 22   41: 21
41: 22   42: 21   42: 22   43: 80   44: 21   44: 22   45: 21   45: 22
46: 21   46: 22   47: 21   47: 22   50: 22   51: 44
There are 23 hits at base#47 These do not occur together.
There are 11 hits at base#44
There are 14 hits at base#22 These do occur together.
There are 9 hits at base#21
BsmAI GTCTCNnnnn                        22
1: 58    3: 58    4: 58    5: 58    8: 58    9: 58
10: 58   13: 70   36: 18   37: 70   38: 70   39: 70
40: 70   41: 70   42: 70   44: 70   45: 70   46: 70
47: 70   48: 48   49: 48   50: 85
There are 11 hits at base#70
-″- Nnnnnngagac                    27
13: 40   15: 48   16: 48   17: 48   18: 48   20: 48
21: 48   22: 48   23: 48   24: 48   25: 48   26: 48
27: 48   28: 48   29: 48   30: 10   30: 48   31: 48
32: 48   33: 48   35: 48   36: 48   43: 40   44: 40
45: 40   46: 40   47: 40
There are 20 hits at base#48
AvaII Ggwcc                     44
Sau96I($M.HaeIII) Ggwcc         44
2: 3     5: 3     6: 44     8: 44     9: 44   10: 44
11: 3     12: 22   13: 22   14: 22   15: 33   15: 47
16: 47   17: 47   18: 47   19: 47   20: 47   21: 47
22: 47   23: 33   23: 47   24: 33   24: 47   25: 33
25: 47   26: 33   26: 47   27: 47   28: 47   29: 47
30: 47   31: 33   31: 47   32: 33   32: 47   33: 33
33: 47   34: 33   34: 47   35: 47   36: 47   37: 47
43: 80   50: 22
There are 23 hits at base#47 44 & 47 never together
There are 4 hits at base#44
PpuMI RGgwccy                    27
6: 43     8: 43     9: 43   10: 43   15: 46   16: 46
17: 46   18: 46   19: 46   20: 46   21: 46   22: 46
23: 46   24: 46   25: 46   26: 46   27: 46   28: 46
30: 46   31: 46   32: 46   33: 46   34: 46   35: 46
36: 46   37: 46   43: 79
There are 22 hits at base#46 43 and 46 never occur together.
There are 4 hits at base#43
BsmFI GGGAC                        3
8: 43   37: 46   50: 77
-″- gtccc                           33
15: 48   16: 48   17: 48     1: 0    1: 0     20: 48
21: 48   22: 48   23: 48   24: 48   25: 48   26: 48
27: 48   28: 48   29: 48   30: 48   31: 48   32: 48
33: 48   34: 48   35: 48   36: 48   37: 54   38: 54
39: 54   40: 54   41: 54   42: 54   43: 54   44: 54
45: 54   46: 54   47: 54
There are 20 hits at base#48
There are 11 hits at base#54
HinfI Ganto                      80
8: 77   12: 16   13: 16   14: 16   15: 16   15: 56
15: 77   16: 16   16: 56   16: 77   17: 16   17: 56
17: 77   18: 16   18: 56   18: 77   19: 16   19: 56
19: 77   20: 16   20: 56   20: 77   21: 16   21: 56
21: 77   22: 16   22: 56   22: 77   23: 16   23: 56
23: 77   24: 16   24: 56   24: 77   25: 16   25: 56
25: 77   26: 16   26: 56   26: 77   27: 16   27: 26
27: 56   27: 77   28: 16   28: 56   28: 77   29: 16
29: 56   29: 77   30: 56   31: 16   31: 56   31: 77
32: 16   32: 56   32: 77   33: 16   33: 56   33: 77
34: 16   35: 16   35: 56   35: 77   36: 16   36: 26
36: 56   36: 77   37: 16   38: 16   39: 16   40: 16
41: 16   42: 16   44: 16   45: 16   46: 16   47: 16
48: 46   49: 46
There are 34 hits at base#16
TfiI Gawtc                        21
8: 77   15: 77   16: 77   17: 77   18: 77   19: 77
20: 77   21: 77   22: 77   23: 77   24: 77   25: 77
26: 77   27: 77   28: 77   29: 77   31: 77   32: 77
33: 77   35: 77   36: 77
There are 21 hits at base#77
MlyI GAGTC                    38
12: 16   13: 16   14: 16   15: 16   16: 16   17: 16
18: 16   19: 16   20: 16   21: 16   22: 16   23: 16
24: 16   25: 16   26: 16   27: 16   27: 26   28: 16
29: 16   31: 16   32: 16   33: 16   34: 16   35: 16
36: 16   36: 26   37: 16   38: 16   39: 16   40: 16
41: 16   42: 16   44: 16   45: 16   46: 16   47: 16
48: 46   49: 46
There are 34 hits at base#16
-″- GACTC                        21
15: 56   16: 56   17: 56   18: 56   19: 56   20: 56
21: 56   22: 56   23: 56   24: 56   25: 56   26: 56
27: 56   28: 56   29: 56   30: 56   31: 56   32: 56
33: 56   35: 56   36: 56
There are 21 hits at base#56
PleI gagtc                           38
12: 16   13: 16   14: 16   15: 16   16: 16   17: 16
18: 16   19: 16   20: 16   21: 16   22: 16   23: 16
24: 16   25: 16   26: 16   27: 16   27: 26   28: 16
29: 16   31: 16   32: 16   33: 16   34: 16   35: 16
36: 16   36: 26   37: 16   38: 16   39: 16   40: 16
41: 16   42: 16   44: 16   45: 16   46: 16   47: 16
48: 46   49: 46
There are 34 hits at base#16
-″- gactc                    21
15: 56   16: 56   17: 56   18: 56   19: 56   20: 56
21: 56   22: 56   23: 56   24: 56   25: 56   26: 56
27: 56   28: 56   29: 56   30: 56   31: 56   32: 56
33: 56   35: 56   36: 56
There are 21 hits at base#56
AlwNI CAGNNNctg                    26
15: 68   16: 68   17: 68   18: 68   19: 68   20: 68
21: 68   22: 68   23: 68   24: 68   25: 68   26: 68
27: 68   28: 68   29: 68   30: 68   31: 68   32: 68
33: 68   34: 68   35: 68   36: 68   39: 46   40: 46
41: 46   42: 46
There are 22 hits at base#69

[0163]

TABLE 255
Analysis of frequency of matching REdaptors in actual V genes
A: HpyCH4V in HC at bases 35-56
Number of mismatches.....................Number
Id	Ntot	0	1	2	3	4	5	6	7	8	9	10	Cut	Id	Probe
1	510	5	11	274	92	61	25	22	11	1	3	5	443	6-1	agttctcccTGCAgctgaactc
2	192	54	42	32	24	15	2	3	10	3	1	6	167	3-11	cactqtatcTGCAaatgaacag
3	58	19	7	17	6	5	1	0	1	0	2	0	54	3-09	ccctgtatcTGCAaatgaacag
4	267	42	33	9	8	8	82	43	22	8	11	1	100	5-51	ccgcctaccTGCAgtggagcag
5	250	111	59	41	24	7	5	1	0	0	2	0	242	3-15	cgctgtatcTGCAaatgaaaag
6	7	0	2	0	1	0	0	0	0	0	4	0	3	7-4.1	cggcatatcTGCAgatctgcag
7	7	0	2	2	0	0	2	1	0	0	0	0	4	3-73	cggcgtatcTGCAaatgaacag
8	26	10	4	1	3	1	2	1	3	1	0	0	19	5-a	ctgcctaccTGCAgtggagcag
9	21	8	2	3	1	6	1	0	0	0	0	0	20	3-49	tcgcctatcTGCAaatgaacag
	1338	249	162	379	149	103	120	71	47	13	23	12	1052
		249	411	790	939		1162		1280		1316
						1042		1233		1293		1338+TZ,1 59
Id	Probe	dotted probe+TZ,1 59
6-1	agttctcccTGCAgctgaactc	agttctcccTGCAgctgaactc
3-11	cactgtatcTGCAaatgaacag	cac.g.at.....aa.....ag
3-09	ccctgtatcTGCAaatgaacag	ccc.g.at.....aa.....ag
5-51	ccgcctaccTGCAgtggagcag	ccgc..a.......tg..g.ag
3-15	cgctgtatcTGCAaatgaacag	c.c.g.at.....aa.....ag
7-4.1	cggcatatcTGCAqatctgcag	c.gca.at......a.ctg.ag
3-73	cggcgtatcTGCAaatgaacag	c.gcg.at.....aa.....ag
5-a	ctgcctaccTGCAgtggagcag	ctgc..a.......tg..g.ag
3-49	tcgcctatcTGCAaatgaacag	tcgc..at.....aa.....ag+TZ,1 59
Seqs with the expected RE site only.......1004
(Counts only cases with 4 or fewer mismatches)
Seqs with only an unexpected site......... 0
Seqs with both expected and unexpected.... 48
(Counts only cases with 4 or fewer mismatches)
Seqs with no sites........................ 0
B: BlpI in HC
Id	Ntot	0	1	2	3	4	5	6	7	8	Ncut	Name
/1	133	/73	/16	/11	/13	//6	//9	//1	//4	//0	119	1-58	acatggaGCTGAGCagactgag
/2	/14	/11	//1	//0	//0	//0	//0	//1	//0	//1	//12	1-02	acatggagctgagcaggctgag
/3	/34	/17	//8	// 2	//6	//1	//0	//0	//0	//0	//0	1-18	acatggagctgaggagcctgag
/4	120	/50	/32	/16	/10	//9	//1	//1	//1	//0	//2	5-51	acctgcagtggagcagcctgaa
/5	/55	/13	/11	/10	/17	//3	//1	//0	//0	//0	//0	3-15	atctgcaaatgaacagcctgaa
/6	340	186	/88	/41	/15	//6	//3	//0	//1	//0	//0	3303	atctgcaaatgaacagcctgag
/7	/82	/25	/16	/25	/12	//1	//3	//0	//0	//0	//0	3-20	atctgcaaatgaacagtctgag
/8	//3	// 0	//2	//0	//1	//0	//0	//0	//0	//0	//0	74.1	atctgcagatctgcagcctaaa
/9	/23	/18	//2	//2	//1	//0	//0	//0	//0	//0	//0	3-66	atcttcaaatgaacagcctgag
10	//2	//1	//0	//1	//0	//0	//0	//0	//0	//0	//0	3-64	atcttcaaatgggcagcctgag
11	486	249	/78	/81	/38	/21	/10	//4	//4	//1	467	4301	ccctgaagctgagctctgtgac
12	/16	//6	//3	//1	//0	//1	//1	//3	//1	//0	//1	6-1	ccctgcagctgaactctgtgac
13	/28	/15	//8	//2	//2	//1	//0	//0	//0	//0	//0	2-70	tccttacaatgaccaacatgga
14	//2	//0	//2	//0	//0	//0	//0	//0	//0	//0	//0	2-26	tccttaccatgaccaacatgga
											601
Name	Full sequence	Dot mode
1-58	acatggaGCTGAGCagcctgag	acatggaGCTGAGCagcctgag
1-02	acatggagctgagcaggctgag	................g.....
1-18	acatggagctgaggagcctgag	.............g........
5-51	acctgcagtggagcagcctgaa	..c..c..tg...........a
3-15	atctgcaaatgaacagcctgaa	.tc..c.aa...a........a
3-30.3	atctgcaaatgaacagcctgag	.tc..c.aa...a.........
3-20	atctgcaaatgaacagtctgag	.tc..c.aa...a...t.....
7-4.1	atctgcagatctgcagcctaaa	.tc..c..a.ct.......a.a
3-66	atcttcaaatgaacagcctgag	.tc.tc.aa...a.........
3-64	atcttcaaatgggcagcctgag	.tc.tc.aa..g..........
4-30.1	ccctgaagctgagctctgtgac	c.c..a........tctg...c
6-1	ccctgcagctgaactctgtgac	c.c..c......a.tctg...c
2-70	tccttacaatgaccaacatgga	t.c.tacaa...c..a.a..ga
2-26	tccttaccatgaccaacatgga	t.c.tacca...c..a.a..ga
Seqs with the expected RE site only....... 597 (counting sequences with 4 or fewer mismatches)
Seqs with only an unexpected site......... 2
Seqs with both expected and unexpected.... 2
Seqs with no sites........................ 686
C: HpyCH4III, Bst4CI, or TaaI in HC
In scoring whether the RE site of interest is present, only ONs that have 4 or fewer mismatches are counted.
Number of sequences.......... 1617
Id	Ntot	0	1	2	3	4	5	6	7	8	Ncut		acngt	acngt
1	244	79	92	43	18	10	1	2	0	0	241	102#1,1	ccgtgtattACTGTgcgagaga	ccgtgtattactgtgcgagaga
2	457	69	150	115	66	34	11	8	3	1	434	103#2,3	ctqtgtattaatgtgcgaqaga	.t....................
3	173	52	45	36	22	14	3	0	0	1	169	108#3	ccgtgtattactgtgcgagagg	.....................g
4	16	0	3	2	2	1	6	0	1	1	8	124#5,1	ccgtgtattactgtgcaacaga	................a.c...
5	4	0	0	1	0	1	1	0	1	0	2	145#6	ccatgtattactgtgcaagata	..a.............a...t.
6	15	1	0	1	0	6	4	1	1	1	8	158#8	ccgtgtattactgtgcggcaga	.................gc...
7	23	4	8	5	2	2	1	1	0	0	21	205#12	ccacatattactgtgcacacag	..aca...........acacag
8	9	1	1	1	0	3	2	1	0	0	6	226#13	ccacatattactgtgcacggat	..aca...........ac.gat
9	7	1	3	1	1	0	0	1	0	0	6	270#14	ccacgtattactgtgcacggat	..ac............ac.gat
10	23	7	3	5	5	2	1	0	0	0	22	309#16,	ccttgtattactgtgcaaaaga	..t.............a.a...
11	35	5	10	7	6	3	3	0	1	0	31	313#18,	ctgtgtattactgtgcaagaga	.t..............a.....
12	18	2	3	2	2	6	1	0	2	0	15	315#19	ccgtgtattactgtaccacaga	..............a.c.c...
13	3	1	2	0	0	0	0	0	0	0	3	320#20	ccttgtatcactgtgcgagaga	..tc.....c............
14	117	29	23	28	22	8	4	2	1	0	110	323#22	ccgtatattactgtgcgaaaga	....a.............a...
15	75	21	25	13	9	1	4	2	0	0	69	330#23,	ctgtgtattactgtgcgaaaga	.t................a...
16	14	2	2	2	3	0	3	1	1	0	9	349#29	ccgtgtattactgtactagaga	..............a.t.....
17	2	0	0	1	0	0	1	0	0	0	1	372#33	ccgtgtattactgtgctagaga	................t.....
18	1	0	0	1	0	0	0	0	0	0	1	373#34	ccgtgtattactgtactagaca	..............a.t...c.
19	2	0	0	0	0	0	0	0	0	2	0	3d#36	ctgtgtattactgtaagaaaga	.t............aa..a...
20	34	4	9	9	4	5	3	0	0	0	31	428#38	ccgtgtattactgtgcgagaaa	....................a.
21	17	5	4	2	2	3	1	0	0	0	16	4302#40	ccgtgtattactgtgccagaga	................c.....
22	75	15	17	24	7	10	1	1	0	0	73	439#44	ctgtgtattactgtgcgagaca	.t..................c.
23	40	14	15	4	5	1	0	1	0	0	39	551#48	ccatgtattactgtgcgagaca	..a.................c.
24	213	26	56	60	42	20	7	2	0	0	204	5a#49	ccatatattactgtgcgagaAA	..a.................AA
Group	337	471	363	218	130	58	23	11	6
Cumulative	337	808	1171	1389	1519	1577	1600	1611	1617
Seqs with the expected RS site only.......1511
Seqs with only an unexpected site......... 0

[0164]

Seqs with both expected and unexpected....8
Seqs with no sites........................0
Analysis repeated using only 8 best REdaptors
Id	Ntot	0	1	2	3	4	5	6	7	8+
1	301	78	101	54	32	16	9	10	1	0	281	102#1	ccgtgtattactgtgcgagaga
2	493	69	155	125	73	37	14	11	3	6	459	103#2	ctgtgtattactgtgcgagaga
3	189	52	45	38	23	18	5	4	1	3	176	108#3	ccgtgtattactgtgcgagagg
4	127	29	23	28	24	10	6	5	2	0	114	323#22	ccgtatattactgtgcgaaaga
5	78	21	25	14	11	1	4	2	0	0	72	330#23	ctgtgtattactgtgcgaaaga
6	79	15	17	25	8	11	1	2	0	0	76	439#44	ctgtgtattactgtgcgagaca
7	43	14	15	5	5	3	0	1	0	0	42	551#48	ccatgtattactgtgcgagaca
8	307	26	63	72	51	38	24	14	13	6	250	5a#49	ccatgtattactgtgcgaga
1	102#1	ccgtgtattactgtgcgagaga ccgtgtattactgtgcgagaga
2	103#2	ctgtgtattactgtgcgagaga    .t....................
3	108#3	ccgtgtattactgtgcgagagg    .....................g
4	323#22	ccgtatattactgtgcgaaaga    ....a.............a...
5	330#23	ctgtgtattactgtgcgaaaga    .t................a...
6	439#44	ctgtgtattactgtgcgagaca    .t.................c.
7	551#48	ccatgtattactgtgcgagaca    ..a.................c.
8	5a#49	ccatgtattactgtgcgagaAA ..a.................AA
Segs with the expected RE site only........1463/1617
Segs with only an unexpected site..........   0
Segs with both expected and unexpected.....   7
Segs with no sites.........................   0

[0165]

TABLE 300
Kappa FR1 GLGs
! 1   2   3   4   5   6   7   8   9   10  11  12
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
! 13  14  15  16  17  18  19  20  21  22  23
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  O12
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  O2
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCG CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  O18
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  O8
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  A20
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  A30
AAC ATC CAG ATG ACC CAG TCT CCA TCT GCC ATG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT  !  L14
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCA CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT  !  L1
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCA CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT  !  L15
GCC ATC CAG TTG ACC CAG TCT CCA TCC TCC CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  L4
GCC ATC CAG TTG ACC CAG TCT CCA TCC TCC CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  L18
GAC ATC CAG ATG ACC CAG TCT CCA TCT TCC GTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT  !  L5
GAC ATC CAG ATG ACC CAG TCT CCA TCT TCT GTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT  !  L19
GAC ATC CAG TTG ACC CAG TCT CCA TCC TTC CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  L8
GCC ATC CGG ATG ACC CAG TCT CCA TTC TCC CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  L23
GCC ATC CGG ATG ACC CAG TCT CCA TCC TCA TTC TCT
GCA TCT ACA GGA GAC AGA GTC ACC ATC ACT TGT  !  L9
GTC ATC TGG ATG ACC CAG TCT CCA TCC TTA CTC TCT
GCA TCT ACA GGA GAC AGA GTC ACC ATC AGT TGT  !  L24
GCC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  L11
GAC ATC CAG ATG ACC GAG TCT CCT TCC ACC CTG TCT  !
GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !  L12
GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC CTG CCC
GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC  !  O11
GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC CTG CCC
GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC  !  O1
GAT GTT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC
GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC  !  A17
GAT GTT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC
GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC  !  A1
GAT ATT GTG ATG ACC CAG ACT CCA CTC TCT CTG TCC
GTC ACC CCT GGA CAG CCG GCC TCC ATC TCC TGC  !  A18
GAT ATT GTG ATG ACC CAG ACT CCA CTC TCT CTG TCC
GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC  !  A2
GAT ATT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC
GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC  !  A19
GAT ATT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC
GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC  !  A3
GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC TCA CCT
GTC ACC CTT GGA GAG CCG GCC TCC ATC TCC TGC  !  A23
GAA ATT GTG TTG ACG CAG TCT CCA GGC ACC CTG TCT
TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !  A27
GAA ATT GTG TTG ACG CAG TCT CCA GCC ACC CTG TCT
TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !  A11
GAA ATA GTG ATG ACG CAG TCT CCA GCC ACC CTG TCT
GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !  L2
GAA ATA GTG ATG ACG CAG TCT CCA GCC ACC CTG TCT
GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !  L16
GAA ATT GTC TTG ACA CAG TCT CCA GCC ACC CTG TCT
TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !  L6
GAA ATT GTG TTG ACA CAG TCT CCA GCC ACC CTG TCT
TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TCG  !  L20
GAA ATT GTA ATG ACA CAG TCT CCA GCC ACC CTG TCT
TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !  L25
GAC ATC GTG ATG ACC CAG TCT CCA GAC TCC CTG GCT
GTG TCT CTG GGC GAG AGG GCC ACC ATC AAC TGC  !  B3
GAA ACG ACA CTC ACG CAG TCT CCA GCA TTC ATG TCA
GCG ACT CCA GGA GAC AAA GTC AAC ATC TCC TGC  !  B2
GAA ATT GTG CTG ACT CAG TCT CCA GAC TTT GAG TCT
GTG ACT CCA AAG GAG AAA GTC ACC ATC ACC TGC  !  A26
GAA ATT GTG CTG ACT CAG TCT CCA GAC TTT CAG TCT
GTG ACT CCA AAG GAG AAA GTC ACC ATC ACC TGC  !  A10
GAT GTT GTG ATG ACA CAG TCT CCA GCT TTC CTC TCT
GTG ACT CCA GGG GAG AAA GTC ACC ACG ACC TGC  !  A14

[0166]

TABLE 302
RERS sites found in Human Kappa FR1 GLGs
			FokI					HPyCH
		MslI	→ ← →	PflFI	BsrI	BsmAI	MnlI	4V
VKI
O12	1-69	3	3		23	12	49		15	18		47	26			36
O2	101-169	103	103		123	112	149		115	118		147	126			136
O18	201-269	203	203		223	212	249		215	218		247	226			236
O8	301-369	303	303		323	312	349		315	318		347	326			336
A20	401-469	403	403		423	412	449		415	418		447	426			436
A30	501-569	503	503		523	512	549		515	518		547	526			536
L14	601-669	603	603			612	649		615	618		647	—			636
L1	701-769	703	703		723	712	749		715	718		747	726			736
L15	801-869	803	803		823	812	849		815	818		847	826			836
L4	901-969	—	903		923	912	949	906	915	918		947	926			936
L18	1001-1069	—	1003			1012	1049	1006	1015	1018		1047	1026			1036
L5	1101-1169	1103	—			1112	1149		1115	1118		1147	—			1136
L19	1201-1269	1203	1203			1212	1249		1215	1218		1247	—			1236
L8	1301-1369	—	1303		1323	1312	1349	1306	1315	1318		1347	—			1336
L23	1401-1469	1403	1403	1408		1412	1449		1415	1418		1447	—			1436
L9	1501-1569	1503	1503	1508	1523	1512	1549		1515	1518		1547	1526			1536
L24	1601-1669	1603		1608	1623	1612	1649		1615	1618		1647	—			1636
L11	1701-1769	1703	1703		1723	1712	1749		1715	1718		1747	1726			1736
L12	1801-1869	1803	1803			1812	1849		1815	1818		1847	—			1836
VKII
O11	1901-1969	—	—			—		—		—					1956	—
O1	2001-2069	—	—			—		—								2056	—
A17	2101-2169	—	—			2112		—		2118					2156	—
A1	2201-2269	—	—			2212		—		2218					2256	—
A18	2301-2369	—	—			—		—		—					2356	—
A2	2401-2469	—	—			—		—		—					2456	—
A19	2501-2569	—	—			2512		—		2518					2556	—
A3	2601-2669	—	—			2612		—		2618					2656	—
A23	2701-2769	—	—			—		—		—				2729	2756	—
VKIII
A27	2801-2869	—	—			2812		—		2818	2839				—
												2860			—
A11	2901-2969	—	—			2912		—		2918	2939					—
													2960			—
L2	3001-3069	—	—			3012		—		3018	3039					—
								—					3060
L16	3101-3169	—	—			3112		—		3118	3139					—
													3160
L6	3201-3269	—	—			3212	—		3218	3239					—
												3260
L20	3301-3369	—	—			3312		—		3318	3339					—
													3360
L25	3401-3469	—	—			3412		—		3418	3439					—
													3460
VKIV
B3	3501-3569	3503	—			3512		3515		3518	3539					—
													3551<
VKV
B2	3601-3669	—	—				3649	—		3618		3647				—
VKVI
A26	3701-3769	—	—			3712		—		3718						—
A10	3801-3869	—	—			3812		—		3818						—
A14	3901-3969	—	—			3912		—		3918				3930>	—
						MaeIII		HpaII
					MlyI	Tsp45I	HphI	MspI
		SfaNI	SfcI	HinfI	→→ ←	same sites	xx38 xx56 xx62	xx06 xx52
VKI
O12	1-69	37	41			53			53		55		56		—
O2	101-169	137	141			153			153		155		156		—
O18	201-269	237	241			253			253		255		256		—
O8	301-369	337	341			353			353		355		356		—
A20	401-469	437	441			453			453		455		456		—
A30	501-569	537	541			553			553		555		556		—
L14	601-669	637	641			653			653		655		656		—
L1	701-769	737	741			753			753		755		756		—
L15	801-869	837	841			853			853		855		856		—
L4	901-969	937	941			953			953		955		956		—
L18	1001-1069	1037	1041			1053			1053		1055		1056		—
L5	1101-1169	1137	1141			1153			1153		1155		1156		—
L19	1201-1269	1237	1241			1253			1253		1255		1256		—
L8	1301-1369	1337	1341			1353			1353		1355		1356		—
L23	1401-1469	1437	1441			1453			1453		1455		1456		1406
L9	1501-1569	1537	1541			1553			1553		1555		1556		1506
L24	1601-1669	1637	1641			1653			1653		1655		1656
L11	1701-1769	1737	1741			1753			1753		1755		1756
L12	1801-1869	1837	1841			1853			1853		1855		1856
VKII
O11	1901-1969	—	—		1918				1918		1937		1938				1952
O1	2001-2069	—	—		2018				2018		2037		2038				2052
A17	2101-2169	—	—		2112			2112			2137		2138				2152
A1	2201-2269	—	—	2212			2212			2237		2238				2252
A18	2301-2369	—	—		2318			2318		2337		2336				2352
A2	2401-2469	—	—		2418			2418		2437		2436				2452
A19	2501-2569	—	—	2512			2512			2537		2538				2552
A3	2601-2669	—	—	2612			2612			2637		2638				2652
A23	2701-2769	—	—		2718			2718		2737		2731*	2738*		—
VKIII
A27	2801-2669	—	—	—			—							—
A11	2901-2969	—	—	—			—							—
L2	3001-3069	—	—	—			—							—
L16	3101-3169	—	—	—			—							—
L6	3201-3269	—	—	—			—							—
L20	3301-3369	—	—	—			—							—
L25	3401-3469	—	—	—			—							—
VKIV
B3	3501-3569	—	—			3525		3525							—
VKV
B2	3601-3669	—	—			3639			3639						—
VKVI
A26	3701-3769	—	—	3712		3739	3712		3739	3737	3755		3756	3762	—
A10	3801-3869	—	—	3812		3839	3812		3839	3837	3855		3856	3862	—
A14	3901-3969	—	—			3939			3939	3937	3955		3956	3962	—
				BsrFI
				Cac8I
			BpmI	NaeI
	BsaJI	BssKI (NstNI)	xx20 xx41 xx44	NgoMI	HaeII
	xx29 xx42 xx43	xx22 xx30 xx43	→→ ←	V	I	Tsp509I
VKI
O12	1-69	—			—			—			—	—	—
O2	101-169	—			—			—			—	—	—
O18	201-269	—			—			—			—	—	—
O8	301-369	—			—			—			—	—	—
A20	401-469	—			—			—			—	—	—
A30	501-569	—			—			—			—	—	—
L14	601-669	—			—			—			—	—	—
L1	701-769	—			—			—			—	—	—
L15	801-869	—			—			—			—	—	—
L4	901-969	—			—			—			—	—	—
L18	1001-1069	—			—			—			—	—	—
L5	1101-1169	—			—			—			—	—	—
L19	1201-1269	—			—			—			—	—	—
L8	1301-1369	—			—			—			—	—	—
L23	1401-1469	—			—			—			—	—	—
L9	1501-1569	—			—			—			—	—	—
L24	1601-1669	—			—			—			—	—	—
L11	1701-1769	—			—			—			—	—	—
L12	1801-1869	—			—			—			—	—	—
VKII
O11	1901-1969		1942				1943			1944	1951	1954	—
O1	2001-2069		2042				2043			2044	2051	2054	—
A17	2101-2169		2142		—			—			2151	2154	—
A1	2201-2269		2242		—			—			2251	2254	—
A18	2301-2369		2342				2343	—			2351	2354	—
A2	2401-2469		2442				2443	—			2451	2454	—
A19	2501-2569		2542				2543			2544	2551	2554	—
A3	2601-2669		2642				2643			2644	2651	2654	—
A23	2701-2769		2742		—			—			2751	2754	—
VKIII
A27	2801-2869			2843	2822		2843	2820	2841		—	—	2803
A11	2901-2969			2943			2943	2920	2941		—	—	2903
L2	3001-3069			3043			3043		3041		—	—	—
L16	3101-3169			3143			3143	3120	3141		—	—	—
L6	3201-3269			3243			3243	3220	3241		—	—	3203
L20	3301-3369			3343			3343	3320	3341		—	—	—	3303
L25	3401-3469			3443			3443	3420	3441		—	—	3403
VKIV
B3	3501-3569	3529				3530		3520			—	3554
VKV
B2	3601-3669						3643	3620	3641	—	—
VKVI
A26	3701-3769				—			3720			—	—	3703
A10	3801-3869				—			3820			—	—	3803
A14	3901-3969			3943			3943	3920	3941		—	—	—

[0167]

TABLE 400
Lambda FR1 GLG sequences
! VL1
CAG TCT GTG CTG ACT CAG CCA CCC TCG GTG TCT GAA
GCC CCC AGG CAG AGG GTC ACC ATC TCC TGT !  1a
cag tct gtg gtg acG cag ccG ccc tcA gtg tct gGG
gcc ccA Ggg cag agg gtc acc atc tcc tgC !  1e
cag tct gtg ctg act cag cca ccc tcA gCg tct gGG
Acc ccc Ggg cag agg gtc acc atc tcT tgt !  1c
cag tct gtg ctg act cag cca ccc tcA gCg tct gGG
Acc ccc Ggg cag agg gtc acc atc tcT tgt !  1g
cag tct gtg Ttg acG cag ccG ccc tcA gtg tct gCG
gcc ccA GgA cag aAg gtc acc atc tcc tgC !  1b
! VL2
CAG TCT GCC CTG ACT CAG CCT CCC TCC GCG TCC GGG
TCT CCT GGA CAG TCA GTC ACC ATC TCC TGC !  2c
cag tct gcc ctg act cag cct cGc tcA gTg tcc ggg
tct cct gga cag tca gtc acc atc tcc tgc !  2e
cag tct gcc ctg act cag cct Gcc tcc gTg tcT ggg
tct cct gga cag tcG Atc acc atc tcc tgc !  2a2
cag tct gcc ctg act cag cct ccc tcc gTg tcc ggg
tct cct gga cag tca gtc acc atc tcc tgc !  2d
cag tct gcc ctg act cag cct Gcc tcc gTg tcT ggg
tct cct gga cag tcG Atc acc atc tcc tgc !  2b2
! VL3
TCC TAT GAG CTG ACT CAG CCA CCC TCA GTG TCC GTG
TCC CCA GGA CAG ACA GCC AGC ATC ACC TGC !   3r
tcc tat gag ctg act cag cca cTc tca gtg tcA gtg
Gcc cTG gga cag acG gcc agG atT acc tgT !  3j
tcc tat gag ctg acA cag cca ccc tcG gtg tcA gtg
tcc cca gga caA acG gcc agG atc acc tgc !  3p
tcc tat gag ctg acA cag cca ccc tcG gtg tcA gtg
tcc cTa gga cag aTG gcc agG atc acc tgc !  3a
tcT tCt gag ctg act cag GAC ccT GcT gtg tcT gtg
Gcc TTG gga cag aca gTc agG atc acA tgc !  3l
tcc tat gTg ctg act cag cca ccc tca gtg tcA gtg
Gcc cca gga Aag acG gcc agG atT acc tgT !  3h
tcc tat gag ctg acA cag cTa ccc tcG gtg tcA gtg
tcc cca gga cag aca gcc agG atc acc tgc !  3e
tcc tat gag ctg aTG cag cca ccc tcG gtg tcA gtg
tcc cca gga cag acG gcc agG atc acc tgc !  3m
tcc tat gag ctg acA cag cca Tcc tca gtg tcA gtg
tcT ccG gga cag aca gcc agG atc acc tgc !  V2-19
! VL4
CTG CCT GTG CTG ACT CAG CCC CCG TCT GCA TCT GCC
TTG CTG GGA GCC TCG ATC AAG CTC ACC TGC !  4c
cAg cct gtg ctg act caA TcA TcC tct gcC tct gcT
tCC ctg gga Tcc tcg Gtc aag ctc acc tgc !  4a
cAg cTt gtg ctg act caA TcG ccC tct gcC tct gcc
tCC ctg gga gcc tcg Gtc aag ctc acc tgc !  4b
! VL5
CAG CCT GTG CTG ACT CAG CCA CCT TCC TCC TCC GCA
TCT CCT GGA GAA TCC GCC AGA CTC ACC TGC !  5e
cag Gct gtg ctg act cag ccG Gct tcc CTc tcT gca
tct cct gga gCa tcA gcc agT ctc acc tgc !  5c
cag cct gtg ctg act cag cca Tct tcc CAT tcT gca
tct Tct gga gCa tcA gTc aga ctc acc tgc !  5b
! VL6
AAT TTT ATG CTG ACT CAG CCC CAC TCT GTG TCG GAG
TCT CCG GGG AAG ACG GTA ACC ATC TCC TGC !  6a
! VL7
CAG ACT GTG GTG ACT CAG GAG CCC TCA CTG ACT GTG
TCC CCA GGA GGG ACA GTC ACT CTC ACC TGT !  7a
cag Gct gtg gtg act cag gag ccc tca ctg act gtg
tcc cca gga ggg aca gtc act ctc acc tgt !  7b
! VL8
CAG ACT GTG GTG ACC CAG GAG CCA TCG TTG TCA GTG
TCC CCT GGA GGG ACA GTC ACA CTC ACT TGT !  8a
! VL9
CAG CCT GTG CTG ACT CAG CCA CCT TCT GCA TCA GCC
TCC CTG GGA GCC TCG GTC ACA CTC ACC TGC !  9a
! VL10
CAG GCA GGG CTG ACT CAG CCA CCC TCG GTG TCC AAG
GGC TTG AGA CAG ACC GCC ACA CTC ACC TGC !  10a

[0168]

TABLE 405
RERSs found in human lambda FR1 GLGs
! There are 31 lambda GLGs
MlyI NnnnnnGACTC   25
1: 6	3: 6	4: 6	6: 6	7: 6	8: 6
9: 6	10: 6	11: 6	12: 6	15: 6	16: 6
20: 6	21: 6	22: 6	23: 6	23: 50	24: 6
25: 6	25: 50	26: 6	27: 6	28: 6	30: 6
31: 6
There are 23 hits at base# 6
-“- GAGTCNNNNNn	1
26: 34
MwoI GCNNNNNnngc   20
1: 9	2: 9	3: 9	4: 9	11: 9	11: 56
12: 9	13: 9	14: 9	16: 9	17: 9	18: 9
19: 9	20: 9	23: 9	24: 9	25: 9	26: 9
30: 9	31: 9
There are 19 hits at base# 9
HinfI Gantc          27
1: 12	3: 12	4: 12	6: 12	7: 12	8: 12
9: 12	10: 12	11: 12	12: 12	15: 12	16: 12
20: 12	21: 12	22: 12	23: 12	23: 46	23: 56
24: 12	25: 12	25: 56	26: 12	26: 34	27: 12
28: 12	30: 12	31: 12
There are 23 hits at base# 12
PleI gactc          25
1: 12	3: 12	4: 12	6: 12	7: 12	8: 12
9: 12	10: 12	11: 12	12: 12	15: 12	16: 12
20: 12	21: 12	22: 12	23: 12	23: 56	24: 12
25: 12	25: 56	26: 12	27: 12	28: 12	30: 12
31: 12
There are 23 hits at base# 12
-“- gagtc          1
26: 34
DdeI Ctnag          32
1: 14	2: 24	3: 14	3: 24	4: 14	4: 24
5: 24	6: 14	7: 14	7: 24	8: 14	9: 14
10: 14	11: 14	11: 24	12: 14	12: 24	15: 5
15: 14	16: 14	16: 24	19: 24	20: 14	23: 14
24: 14	25: 14	26: 14	27: 14	28: 14	29: 30
30: 14	31: 14
There are 21 hits at base# 14
BsaJI Ccnngg          38
1: 23	1: 40	2: 39	2: 40	3: 39	3: 40
4: 39	4: 40	5: 39	11: 39	12: 38	12: 39
13: 23	13: 39	14: 23	14: 39	15: 38	16: 39
17: 23	17: 39	18: 23	18: 39	21: 38	21: 39
21: 47	22: 38	22: 39	22: 47	26: 40	27: 39
28: 39	29: 14	29: 39	30: 38	30: 39	30: 47
31: 23	31: 32
There are 17 hits at base# 39
There are 5 hits at base# 38
There are 5 hits at base# 40 Makes cleavage
ragged.
MnlI cctc          35
1: 23	2: 23	3: 23	4: 23	5: 23	6: 19
6: 23	7: 19	8: 23	9: 19	9: 23	10: 23
11: 23	13: 23	14: 23	16: 23	17: 23	18: 23
19: 23	20: 47	21: 23	21: 29	21: 47	22: 23
22: 29	22: 35	22: 47	23: 26	23: 29	24: 27
27: 23	28: 23	30: 35	30: 47	31: 23
There are 21 hits at base# 23
There are 3 hits at base# 19
There are 3 hits at base# 29
There are 1 hits at base# 26
There are 1 hits at base# 27 These could make
cleavage ragged.
-“- gagg          7
1: 48	2: 48	3: 48	4: 48	27: 44	28: 44
29: 44
BssKI Nccngg          39
1: 40	2: 39	3: 39	3: 40	4: 39	4: 40
5: 39	6: 31	6: 39	7: 31	7: 39	8: 39
9: 31	9: 39	10: 39	11: 39	12: 38	12: 52
13: 39	13: 52	14: 52	16: 39	16: 52	17: 39
17: 52	18: 39	18: 52	19: 39	19: 52	21: 38
22: 38	23: 39	24: 39	26: 39	27: 39	28: 39
29: 14	29: 39	30: 38
There are 21 hits at base# 39
There are 4 hits at base# 38
There are 3 hits at base# 31
There are 3 hits at base# 40 Ragged
BstNI CCwgg          30
1: 41	2: 40	5: 40	6: 40	7: 40	8: 40
9: 40	10: 40	11: 40	12: 39	12: 53	13: 40
13: 53	14: 53	16: 40	16: 53	17: 40	17: 53
18: 40	18: 53	19: 53	21: 39	22: 39	23: 40
24: 40	27: 40	28: 40	29: 15	29: 40	30: 39
There are 17 hits at base# 40
There are 7 hits at base# 53
There are 4 hits at base# 39
There are 1 hits at base# 41 Ragged
PspGI ccwgg          30
1: 41	2: 40	5: 40	6: 40	7: 40	8: 40
9: 40	10: 40	11: 40	12: 39	12: 53	13: 40
13: 53	14: 53	16: 40	16: 53	17: 40	17: 53
18: 40	18: 53	19: 53	21: 39	22: 39	23: 40
24: 40	27: 40	28: 40	29: 15	29: 40	30: 39
There are 17 hits at base# 40
There are 7 hits at base# 53
There are 4 hits at base# 39
There are 1 hits at base# 41
ScrFI CCngg          39
1: 41	2: 40	3: 40	3: 41	4: 40	4: 41
5: 40	6: 32	6: 40	7: 32	7: 40	8: 40
9: 32	9: 40	10: 40	11: 40	12: 39	12: 53
13: 40	13: 53	14: 53	16: 40	16: 53	17: 40
17: 53	18: 40	18: 53	19: 40	19: 53	21: 39
22: 39	23: 40	24: 40	26: 40	27: 40	28: 40
29: 15	29: 40	30: 39
There are 21 hits at base# 40
There are 4 hits at base# 39
There are 3 hits at base# 41
MaeIII gtnac          16
1: 52	2: 52	3: 52	4: 52	5: 52	6: 52
7: 52	9: 52	26: 52	27: 10	27: 52	28: 10
28: 52	29: 10	29: 52	30: 52
There are 13 hits at base# 52
Tsp45I gtsac          15
1: 52	2: 52	3: 52	4: 52	5: 52	6: 52
7: 52	9: 52	27: 10	27: 52	28: 10	28: 52
29: 10	29: 52	30: 52
There are 12 hits at base# 52
HphI tcacc          26
1: 53	2: 53	3: 53	4: 53	5: 53	6: 53
7: 53	8: 53	9: 53	10: 53	11: 59	13: 59
14: 59	17: 59	18: 59	19: 59	20: 59	21: 59
22: 59	23: 59	24: 59	25: 59	27: 59	28: 59
30: 59	31: 59
There are 16 hits at base# 59
There are 10 hits at base# 53
BspMI ACCTGCNNNNn          14
11: 61	13: 61	14: 61	17: 61	18: 61	19: 61
20: 61	21: 61	22: 61	23: 61	24: 61	25: 61
30: 61	31: 61
There are 14 hits at base# 61 Goes into CDR1

[0169]

TABLE 500
h3401-h2 captured Via CJ with BsmAI
!  1   2   3   4   5   6   7   8   9  10  11  12  13  14  15
!  S   A   Q   D   I   Q   M   T   Q   S   P   A   T   L   S
aGT GCA Caa gac atc cag atg acc cag tct cca gcc acc ctg tct
!  ApaLI...                                 a gcc acc !
L25,L6,L20,L2,L16,A11
!  Extender.................................Bridge...
! 16  17  18  19  20  21  22  23  24  25  26  27  28  29  30
!  V   S   P   G   E   R   A   T   L   S   C   R   A   S   Q
gtg tct cca ggg gaa agg gcc acc ctc tcc tgc agg gcc agt cag
! 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45
!  S   V   S   N   N   L   A   W   Y   Q   Q   K   P   G   Q
agt gtt agt aac aac tta gcc tgg tac cag cag aaa cct ggc cag
! 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60
!  V   P   R   L   L   I   Y   G   A   S   T   R   A   T   D
gtt ccc agg ctc ctc atc tat ggt gca tcc acc agg gcc act gat
! 61  62  63  64  65  66  67  68  69  70  71  72  73  74  75
!  I   P   A   R   F   S   G   S   G   S   G   T   D   F   T
atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc act
! 76  77  78  79  80  81  82  83  84  85  86  87  88  89  90
!  L   T   I   S   R   L   E   P   E   D   F   A   V   Y   Y
ctc acc atc agc aga ctg gag cct gaa gat ttt gca gtg tat tac
! 91  92  93  94  95  96  97  98  99 100 101 102 103 104 105
!  C   Q   R   Y   G   S   S   P   G   W   T   F   G   Q   G
tgt cag cgg tat ggt agc tca ccg ggg tgg acg ttc ggc caa ggg
! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
!  T   K   V   E   I   K   R   T   V   A   A   P   S   V   F
acc aag gtg gaa atc aaa cga act gtg gct gca cca tct gtc ttc
! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
!  I   F   P   P   S   D   E   Q   L   K   S   G   T   A   S
atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct
! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150
!  V   V   C   L   L   N   N   F   Y   P   R   E   A   K   V
gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta
! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
!  Q   W   K   V   D   N   A   L   Q   S   G   N   S   Q   E
cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag
! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
!  S   V   T   E   Q   D   S   K   D   S   T   Y   S   L   S
agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc
! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
!  S   T   L   T   L   S   K   A   D   Y   E   K   H   K   V
agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc
! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
!  Y   A   C   E   V   T   H   Q   G   L   S   S   P   V   T
tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg cct gtc aca
! 211 212 213 214 215 216 217 218 219 220 221 222 223
!  K   S   F   N   K   G   E   C   K   G   E   F   A
aag agc ttc aac aaa gga gag tgt aag ggc gaa ttc gc.....

[0170]

TABLE 501
h3401-d8 KAPPA captured with CJ and BsmAI
!  1   2   3   4   5   6   7   8   9  10  11  12  13  14  15
!  S   A   Q   D   I   Q   M   T   Q   S   P   A   T   L   S
aGT GCA Caa gac atc cag atg acc cag tct cct gcc acc ctg tct
!  ApaLI...Extender.........................a gcc acc !
L25,L6,L20,L2,L16,A11
!                                           A GCC ACC CTG TCT !L2
!  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30
!  V   S   P   G   E   R   A   T   L   S   C   R   A   S   Q
gtg tct cca ggt gaa aga gcc acc ctc tcc tgc agg gcc agt cag
! GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !     L2
!  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45
!  N   L   L   S   N   L   A   W   Y   Q   Q   K   P   G   Q
aat ctt ctc agc aac tta gcc tgg tac cag cag aaa cct ggc cag
!  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60
!  A   P   R   L   L   I   Y   G   A   S   T   G   A   I   G
gct ccc agg ctc ctc atc tat ggt gct tcc acc ggg gcc att ggt
!  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75
!  I   P   A   R   F   S   G   S   G   S   G   T   E   F   T
atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gag ttc act
!  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90
!  L   T   I   S   S   L   Q   S   E   D   F   A   V   Y   F
ctc acc atc agc agc ctg cag tct gaa gat ttt gca gtg tat ttc
!  91  92  93  94  95  96  97  98  99 100 101 102 103 104 105
!  C   Q   Q   Y   G   T   S   P   P   T   F   G   G   G   T
tgt cag cag tat ggt acc tca ccg ccc act ttc ggc gga ggg acc
! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
!  K   V   E   I   K   R   T   V   A   A   P   S   V   F   I
aag gtg gag atc aaa cga act gtg gct gca cca tct gtc ttc atc
! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
!  F   P   P   S   D   E   Q   L   K   S   G   T   A   S   V
ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt
! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150
!  V   C   P   L   N   N   F   Y   P   R   E   A   K   V   Q
gtg tgc ccg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag
! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
!  W   K   V   D   N   A   L   Q   S   G   N   S   Q   E   S
tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt
! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
!  V   T   E   Q   D   N   K   D   S   T   Y   S   L   S   S
gtc aca gag cag gac aac aag gac agc acc tac agc ctc agc agc
! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
!  T   L   T   L   S   K   V   D   Y   E   K   H   E   V   Y
acc ctg acg ctg agc aaa gta gac tac gag aaa cac gaa gtc tac
! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
!  A   C   E   V   T   H   Q   G   L   S   S   P   V   T   K
gcc tgc gaa gtc acc cat cag ggc ctt agc tcg ccc gtc acg aag
! 211 212 213 214 215 216 217 218 219 220 221 222 223
!  S   F   N   R   G   E   C   K   K   E   F   V
agc ttc aac agg gga gag tgt aag aaa gaa ttc gtt t

[0171]

TABLE 508
Human heavy chains bases 88.1 to 94.2
Number of sequences..........   840
	Number of Mismatchers.........		Probe
Id	Ntot	0	1	2	3	4	5	6	7	Name	Sequence............	Dot form............
1	364	152	97	76	26	7	4	2	0	VHS881-1.1	gctgtgtattactgtgcgag	gctgtgtattactgtgcgag
2	265	150	60	33	13	5	4	0	0	VHS881-1.2	gccgtgtattactgtgcgag	..c.................
3	96	14	34	16	10	5	7	9	1	VHS881-2.1	gccgtatattactgtgcgag	..c..a..............
4	20	0	3	4	9	2	2	0	0	VHS881-4.1	gccgtgtattactgtacgag	..c............a....
5	95	25	36	18	11	2	2	0	1	VHS881-9.1	gccatgtattactgtgcgag	..ca................
	840	341	230	147	69	21	19	11	2
		341	571	718	787	808	827	838	840
	88 89 90 91 92 93 94 95 Codon number as in Table 195
	Recognition........... Stem...... Loop. Stem......
(VHS881-1.1)	5′-gctgtgtat|tact-gtgcgag cAcATccgTg TTgTT cAcggATgTg-3′
(VHS881-1.2)	5′-gccgtgtat|tact-gtgcgag cAcATccgTg TTgTT cAcggATgTg-3′
(VHS881-2.1)	5′-gccgtatat|tact-gtgcgag cAcATccgTg TTgTT cAcggATgTg-3′
(VHS881-4.1)	5′-gccgtgtat|tact-gtacgag cAcATccgTg TTgTT cAcggATgTg-3′
(VHS881-9.1)	5′-gccatgtat|tact-gtgccgagcAcATccgTg TTgTT cAcggATgTg-3′
	| site of substrate cleavage
(FOKIact)	5′-cAcATccgTg TTgTT cAcggATgTg-3′
(VHEx881)	5′-AATAgTAgAc TgcAgTgTcc TcAgcccTTA AgcTgTTcAT cTgcAAgTAg-
	AgAgTATTcT TAgAgTTgTc TcTAgAcTTA gTgAAgcg-3′
! note that VHEx881 is the reverse complement of the ON below
!	[RC] 5′-cgCttcacTaag-
!	Scab........
!	Synthetic 3-23 as in Table 206
!	|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
!	XbaI...
!	|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|t-3′
!	AflII...
(VHBA881)	5′-cgCttcacTaag-
	|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
	|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgt gcg ag-3′
(VHBB881)	5′-cgCttcacTaag-
	TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
	|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgt Acg ag-3′
(VH881PCR)	5′-cgCttcacTaag|TCT|AGA|gac|aac-3′

[0172]

TABLE 512

Kappa, bases 12-30

!

ID

Ntot

0

1

2

3

4

5

6

Name

Sequence..........

Dot Form...........

!

1

84

40

21

20

1

2

0

0

SK12O12

gacccagtctccatcctcc

gacccagtctccatcctcc

!

2

32

19

3

6

2

1

0

1

SK12A17

gactcagtctccactctcc

..t..........ct....

!

3

26

17

8

1

0

0

0

0

SK12A27

gacgcagtctccaggcacc

..g..........gg.a..

!

4

40

21

18

1

0

0

0

0

SK12A11

gacgcagtctccagccacc

..g..........g..a..

!

182

97

50

28

3

3

0

1

!

97

147

175

178

181

181

182

URE adapters:

!

Stem...... Loop. Stem...... Recognition........

(SzKB1230-012)

5′-cAcATccgTg TTgTT cAcggATgTg ggAggATggAgAcTgggTc-3′

!

[RC] 5′-gacccagtctccatcctcc cAcATccgTg AAcAA cAcggATgTg-3′

!

Recognition........ Stem...... loop. Stem......

!

FokI.            FokI.

!

!

Stem...... Loop. Stem...... Recognition........

(SzKB1230-A17)

5′-cAcATccgTg TTgTT cAcggATgTg ggAgAgTggAgAcTgAgTc-3′

!

[RC] 5′-gactcagtctccactctcc cAcATccgTg AAcAA cAcggATgTg-3′

!

Recognition........ Stem...... loop. Stem......

!

FokI.            FokI.

!

!

Stem...... Loop. Stem...... Recognition........

(SzKB1230-A27)

5′-cAcATccgTg TTgTT cAcggATgTg ggTgccTggAgAcTgcgTc-3′

!

[RC] 5′-gacgcagtctccaggcacc cAcATccgTg AAcAA cAcggATgTg-3′

!

Recognition........ Stem...... loop. Stem......

!

FokI.            FokI.

!

!

Stem...... Loop. Stem...... Recognition........

(SzKB1230-All)

5′-cAcATccgTg TTgTT cAcggATgTg ggTggcTggAgAcTgcgTc-3′

!

[RC[ 5′-gacgcagtctccagccacc cAcATccgTg AAcAA cAcggATgTg-3′

!

Recognition........ Stem...... loop. Stem......

!

FokI.            FokI.

What happens in the upper strand:

(SzKB1230-012*)

5′-gac cca gtc|tcc a-tc ctc c-3′

!

| Site of cleavage in substrate

!

(SzKB1230-A17*)

5′-gac tca gtc|tcc a-ct ctc c-3′

!

(SzKB1230-A27*)

5′-gac gca gtc|tcc a-gg cac c-3′

!

(SzKB1230-A11*)

5′-gac gca gtc|tcc a-gc cac c-3′

!

(kapextURE)

5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg-3′

!sense strand

Scab.............ApaLI.

(kapextUREPCR)

5′-ccTctactctTgTcAcAgTg-3′

Scab.............

(kaBRO1UR)

5′-ggAggATggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′

!

[RC]5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-tc ctc c-3′ ON above is R C. of

this one

(kaBRO2UR)

5′-ggAgAgTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′

!

[RC]5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-ct ctc c-3′ ON above is R.C. of

this one

(kaBRO3UR)

5′-ggTgccTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′

!

[RC]5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-gg cac c-3′ ON above is R.C. of

this one

(kaBRO4UR)

5′-ggTggcTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′

!

[RC]5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-gc cac c-3′ ON above is R.C. of

this one

Scab.............ApaLI.

|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-

|aac|aqC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgt Acg ag-3′

(VH881PCR)

5′-cgCttcacTaag|TCT|AGA|gac|aac-3′

[0173]

TABLE 512

Kappa, bases 12-30

!

!

ID

Ntot

0

1

2

3

4

5

6

Name

Sequence...........

Dot Form...........

!

1

84

40

21

20

1

2

0

0

SK12012

gacccagtctccatcctcc

gacccagtctccatcctcc

!

2

32

19

3

6

2

1

0

1

SK12A17

gactcagtctccactctcc

..t..........ct....

!

3

26

17

8

1

0

0

0

0

SK12A27

gacgcagtctccaggcacc

...g.........gg.a..

!

4

40

21

18

1

0

0

0

0

SK12A11

gacgcagtctccagccacc

...g.........g..a..

!

182

97

50

28

3

3

0

1

!

97

147

175

178

181

181

182

!

URE adapters:

!

Stem...... Loop. Stem...... Recognition........

(SzKB1230-012)

5′-cAcATccgTg TTgTT cAcggATgTg ggAggATggAgAcTgggTc-3′

!

[RC] 5′-gacccagtctccatcctcc cAcATccgTg AAcAA cAcggATgTg-3′

!

Recognition........ Stem...... loop. Stem......

FokI.            FokI.

!

!

Stem...... Loop. Stem...... Recognition........

(SzKB1230-A17)

5′-cAcATccgTg TTgTT cAcggATgTg ggAgAgTggAgAcTgAgTc-3′

!

[RC] 5′-gactcagtctccactctcc cAcATccgTg AAcAA cAcggATqTg-3′

!

Recognition........ Stem...... loop. Stem......

FokI.            FokI.

!

Stem...... Loop. Stem...... Recognition........

(SzKB1230-A27)

5′-cAcATccgTg TTgTT cAcggATgTg ggTgccTggAgAcTgcgTc-3′

!

[RC] 5′-gacgcagtctccaggcacc cAcATccgTg AAcAA cAcggATgTg-3′

Recognition........ Stem...... loop. Stem......

FokI.            FokI.

Stem...... Loop. Stem...... Recognition........

(SzKB1230-A11)

5′-cAcATccgTg TTgTT cAcggATgTg ggTggcTggAgAcTgcgTc-3′

[RC] 5′-gacgcagtctccagccacc cAcATccgTg AAcAA cAcggATgTg-3′

Recognition........ Stem...... loop. Stem......

FokI.            FokI.

What happens in the upper strand:

(SzKB1230-012*)

5′-gac cca gtc|tcc a-tc ctc c-3′

!

| Site of cleavage in substrate

!

(SzKB1230-A17*)

5′-gac tca gtc|tcc a-ct ctc c-3′

!

(SzKB1230-A27*)

5′-gac gca gtc|tcc a-gg cac c-3′

!

(SzKB1230-A11*)

5′-gac gca gtc|tcc a-gc cac c-3′

(kapextURE)

5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg-3′

!sense strand

Scab.............ApaLI.

(kapextUREPGR)

5′-ccTctactctTgTcAcAgTg-3′

Scab.............

(kaBRO1UR)

5′-ggAggATggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′

!

[RC] 5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-tc ctc c-3′ ON above is R.C. of this one

(kaBRO2UR)

5′-ggAgAgTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′

!

[RC] 5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-ct ctc c-3′ ON above is R.C. of this one

(kaBRO3UR)

5′-ggTgccTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′

[RC] 5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-gg cac c-3′ ON above is R C. of this one

(kaBRO4UR)

5′-ggTggcTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′

!

[RC] 5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-gc cac c-3′ ON above is R.C. of this one

Scab.............ApaLI.

[0174]

TABLE 515

Lambda URE adapters bases 13.3 to 19.3

!

!

Number of sequences.......... 128

!

!

Number of mismatches..............

!

Id

Ntot

0

1

2

3

4

5

6

7

8

Name

Sequence...........

Dot form...........

!

1

58

45

7

1

0

0

0

2

2

1

VL133-2a2

gtctcctggacagtcgatc

gtctcctggacagtcgatc

!

2

16

10

1

0

1

0

1

1

0

2

V1133-31

ggccttgggacagacagtc

.g.cttg......a.ag..

!

3

17

6

0

0

0

4

1

1

5

0

VL133-2c

gtctcctggacagtcagtc

...............ag..

!

4

37

3

0

10

4

4

3

7

4

2

VL133-1c

ggccccagggcagagggtc

.g.c..a..g...ag.g..

!

128

64

8

11

5

8

5

11

11

5

!

64

72

83

88

96

101

112

123

128

!

!

Stem...... loop. Stem...... Recognition........

(VL133-2a2)

5′-cAcATccgTg TTgTT cAcggATgTg gATcgAcTgTccAggAgAc-3′

!

[RC] 5′-gtctcctggacagtcgatc cAcATccgTg AAcAA cAcggATgTg-3′

Recognition........ Stem...... Loop. Stem......

!

!

Stem...... loop. Stem...... Recognition........

(VL133-31)

5′-cAcATccgTg TTgTT cAcggATgTg gAcTgTcTgTcccAAggcc-3′

!

[RC] 5′-ggccttgggacagacagtc cAcATccgTg AAcAA cAcggATgTg-3′

Recognition........ Stem...... Loop. Stem......

!

!

Stem...... loop. Stem...... Recognition........

(VL133-2c)

5′-cAcATccgTg TTgTT cAcggATgTg gAcTgAcTgTccAggAgAc-3′

[RC] 5′-gtctcctggacagtcagtc cAcATccgTg AAcAA cAcggATgTg-3′

!

Recognition........ Stem...... Loop. Stem......

!

!

Stem...... loop. Stem...... Recognition........

(VL133-1c)

5′-cAcATccgTg TTgTT cAcggATgTg gAcccTcTgcccTggggcc-3′

[RC] 5′-ggccccagggcagagggtc cAcATccgTg AAcAA cAcggATgTg-3′

What happens in the top strand:

!

|site of cleavage in the upper strand

(VL133-2a2*)

5′-g tct cct g|ga cag tcg atc

!

(VL133-31*)

5′-g gcc ttg g|ga cag aca gtc

!

(VL133-2c*)

5′-g tct cct g|ga cag tca gtc

!

(VL133-1c*)

5′-g gcc cca g|gg cag agg gtc

!

! The following Extenders and Bridges all encode the AA sequence of 2a2 for

codons 1-15

!

1

(ON_LamEx133)

5′-ccTcTgAcTgAgT gcA cAg -

!

!

2   3   4   5   6   7   8   9   10  11  12

AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT-

!

!

13  14  15

tcC ccG g !  2a2

!                       1

(ON_LamB1-133)

[RC] 5′-ccTcTgAcTgAgT gcA cAg -

!

!

2   3   4   5   6   7   8   9   10  11  12

AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT-

!

!

13  14  15

!

tcC ccG g ga cag tcg at-3′ ! 2a2 N.B. the actual seq is the

reverse complement of the

one shown.

(ON_LamB2-133)

[RC] 5′-ccTcTgAcTgAgT gcA cAg -

!

2   3   4   5   6   7   8   9   10  11  12

AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT-

!

13  14  15

tcC ccG g ga cag aca gt-3′ ! 31 N.B. the actual seq. is the

!

reverse complement of the

!

one shown.

!

(ON_LamB3-133)

[RC] 5′-ccTcTgAcTgAgT gcA cAg -

!

2   3   4   5   6   7   8   9   10  11  12

AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT-

!

13  14  15

tcC ccG g ga cag tca gt -3′! 2c N.B. the actual seq is the

reverse complement of the

one shown.

(ON_LamB4-133)

[RC] 5′-ccTcTgAcTgAgT gcA cAg -

!

2   3   4   5   6   7   8   9   10  11  12

AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT-

!

13  14  15

tcC ccG g gg cag agg gt-3′ ! 1c N.B. the actual seq is the

reverse complement of the

one shown.

(ON_Lam133PCR)

5′-ccTcTgAcTgAgT gcA cAg AGt gc-3′

[0175]

TABLE 525
ONs used in Capture of kappa light chains using CJ
method and BsmAI
All ONs are written 5′ to 3′.
REdapters (6)
ON_20SK15012
gggAggATggAgAcTgggTc
ON_20SK15L12
gggAAgATggAgAcTgggTc
ON_20SK15A17
gggAgAgTggAgAcTgAgTc
ON_20SK15A27
gggTgccTggAgAcTgcgTc
ON_20SK15A11
gggTggcTggAgAcTgcgTc
ON_20SK15B3
gggAgTcTggAgAcTgggTc
Bridges (6)
kapbri1012
gggAggATggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbri1L12
gggAAgATggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbri1A17
gggAgAgTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbri1A27
gggTgccTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbri1A11
gggTggcTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbri1B3
gggAgTcTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
Extender (5′biotinylated)
kapext1bi
ccTcTgTcAcAgTgcAcAAgAcATccAgATgAcccAgTcTcc
Primers
kaPCRt1
ccTcTgTcAcAgTgcAcAAgAc
kapfor
5′-aca ctc tcc cct gtt gaa gct ctt-3′

[0176]

	TABLE 530
	PCR program for
	amplification of kappa DNA
	95° C.	5	minutes
	95° C.	15	seconds
	65° C.	30	seconds
	72° C.	1	minute
	72° C.	7	minutes
	4° C.		hold
	Reagents (100 ul reaction):
	Template	50	ng
	10x turbo PCR buffer	1x
	turbo Pfu	4U
	dNTPs	200	μM each
	kaPCRt1	300	nM
	kapfor	300	nM

[0177]

TABLE 610
Stuffer used in VH
1   TCCGGAGCTT CAGATCTGTT TGCCTTTTTG TGGGGTGGTG CAGATCGCGT TACGGAGATC
61  GACCGACTGC TTGAGCAAAA GCCACGCTTA ACTGCTGATC AGGCATGGGA TGTTATTCGC
121 CAAACCAGTC GTCAGGATCT TAACCTGAGG CTTTTTTTAC CTACTCTGCA AGCAGCGACA
181 TCTGGTTTGA CACAGAGCGA TCCGCGTCGT CAGTTGGTAG AAACATTAAC ACGTTGGGAT
241 GGCATCAATT TGCTTAATGA TGATGGTAAA ACCTGGCAGC AGCCAGGCTC TGCCATCCTG
301 AACGTTTGGC TGACCAGTAT GTTGAAGCGT ACCGTAGTGG CTGCCGTACC TATGCCATTT
361 GATAAGTGGT ACAGCGCCAG TGGCTACGAA ACAACCCAGG ACGGCCCAAC TGGTTCGCTG
421 AATATAAGTG TTGGAGCAAA AATTTTGTAT GAGGCGGTGC AGGGAGACAA ATCACCAATC
481 CCACAGGCGG TTGATCTGTT TGCTGGGAAA CCACAGCAGG AGGTTGTGTT GGCTGCGCTG
541 GAAGATACCT GGGAGACTCT TTCCAAACGC TATGGCAATA ATGTGAGTAA CTGGAAAACA
601 CCTGCAATGG CCTTAACGTT CCGGGCAAAT AATTTCTTTG GTGTACCGCA GGCCGCAGCG
661 GAAGAAACGC GTCATCAGGC GGAGTATCAA AACCGTGGAA CAGAAAACGA TATGATTGTT
721 TTCTCACCAA CGACAAGCGA TCGTCCTGTG CTTGCCTGGG ATGTGGTCGC ACCCGGTCAG
781 AGTGGGTTTA TTGCTCCCGA TGGAACAGTT GATAAGCACT ATGAAGATCA GCTGAAAATG
841 TACGAAAATT TTGGCCGTAA GTCGCTCTGG TTAACGAAGC AGGATGTGGA GGCGCATAAG
901 GAGTCGTCTA GA

[0178]

TABLE 620
DNA sequence off pCEs5
!	pCES5 6680 bases=pCes4 with stuffers in CDR1-2 and CDR3 2000.12.13
!
!	Ngene=6680
!	Useful REs (cut MAnoLI fewer than 3 times) 2000.06.05
!
!	Non-cutters
!	Acc65I Ggtacc	AfeI AGCgct	AvrII Cctagg
!	BsaBI GATNNnnatc	BsiWI Cqtacg	BsmFI Nnnnnnnnnnnnnnngtccc
!	BsrGI Tgtaca	BstAPI GCANNNNntgc	BstBI TTcgaa
!	Bst817I GTAtac	BtrI CACgtg	Ecl136I GAGctc
!	EcoRV GATatc	FseI GGCCGGcc	KpnI GGTACc
!	MscI TGGcca	NruI TCGcga	NsiI ATGCAt
!	PacI TTAATtaa	PmeI GTTTaac	PmlI CACgtg
!	PpuMT RGgwccy	PshAI GACNNnngtc	SacI GAGCTc
!	SacII CCGCgg	SbfI CCTGCAgg	SexAI Accwggt
!	SgfI GCGATcgc	SnaBI TACgta	SpeI Actagt
!	SphI GCATGc	Sse8387I CCTGCAgg	StuI AGGcct
!	SwaI ATTTaaat	XmaI Cccggg
!
!	cutters
!	Enzymes that cut more than	3 times.
!	AlwNI CAGNNNctg	5
!	BsgI ctgcac	4
!	BsrFI Rccggy	5
!	EarI CTCTTCNnnn	4
!	FauI nNNNNNNGCGGG	10
!
!	Enzymes that cut from 1 to	3 times.
!
!	Eco0109I RGgnccy	3	7	2636	4208
!	BssSI Ctcgtg	1	12
!	-″- Cacgag	1	1703
!	BspHI Tcatga	3	43	148	1156
!	AatII GACGTc	1	65
!	BciVI GTATCCNNNNNN	2	140	1667
!	Eco57I CTGAAG	1	301
!	-″- cttcag	2	1349
!	AvaI Cycgrg	3	319	2347	6137
!	BsiHKAI GWGCWc	3	401	2321	4245
!	HgiAI GWGCWc	3	401	2321	4245
!	BcgI gcannnnnntcg	1	461
!	ScaI AGTact	1	505
!	PvuI CGATcg	3	616	3598	5926
!	FspI TGCgca	2	763	5946
!	BglI GCCNNNNnggc	3	864	2771	5952
!	BpmI CTGGAG	1	898
!	-″- ctccag	1	4413
!	BsaI GGTCTCNnnnn	1	916
!	AhdI GACNNNnngtc	1	983
!	Eam1105I GACNNNnngtc	1	983
!	DrdI GACNNNNnngtc	3	1768	6197	6579
!	SapI gaagagc	1	1998
!	PvuII CAGctg	3	2054	3689	5896
!	PflMT CCANNNNntgg	3	2233	3943	3991
!	HindIII Aagctt	1	2235
!	ApaLI Gtgcac	1	2321
!	BspMI Nnnnnnnnngcaggt	1	2328
!	-″- ACCTGCNNNNn	2	3460
!	PstI CTGCAg	1	2335
!	AccI GTmkac	2	2341	2611
!	HincII GYYrac	2	2341	3730
!	SalI Gtcgac	1	2341
!	TliI Ctcgag	1	2347
!	XhoI Ctcgag	1	2347
!	BbsI gtcttc	2	2383	4219
!	BlpI GCtnagc	1	2580
!	EspI GCtnagc	1	2580
!	SgrAI CRccggyg	1	2648
!	AgeI Accggt	2	2649	4302
!	AscI GGcgcgcc	1	2689
!	BssHII Gcgcgc	1	2690
!	SfiI GGCCNNNNnggcc	1	2770
!	NaeI GCCggc	2	2776	6349
!	NgoMIV Gccggc	2	2776	6349
!	BtgI Ccrygg	3	2781	3553	5712
!	DsaI Ccrygg	3	2781	3553	5712
!	NcoI Ccatgg	1	2781
!	StyI Ccwwgg	3	2781	4205	4472
!	MfeI Caattg	1	2795
!	BspEI Tccgga	1	2861
!	BglII Agatct	1	2872
!	BclI Tgatca	1	2956
!	Bsu36I CCtnagg	3	3004	4143	4373
!	XcmI CCANNNNNnnnntgg	1	3215
!	MluI Acgcgt	1	3527
!	HpaI GTTaac	1	3730
!	XbaI Tctaga	1	3767
!
!	AflII Cttaag	1	3811
!	BsmI NGcattc	1	3821
!	-″- GAATGCN	1	4695
!	RsrII CGgwccg	1	3827
!	NheI Gctagc	1	4166
!	BstEII Ggtnacc	1	4182
!	BsmBI CGTCTCNnnnn	2	4188	6625
!	-″- Nnnnnngagacg	1	6673
!	ApaI GGGCCc	1	4209
!	BanII GRGCYc	3	4209	4492	6319
!	Bsp120I Gggccc	1	4209
!	PspOMI Gggccc	1	4209
!	BseRI NNnnnnnnnnctcctc	1	4226
!	-″- GAGGAGNNNNNNNNNN	1	4957
!	EcoNI CCTNNnnnagg	1	4278
!	PflFI GACNnngtc	1	4308
!	TthlllI GACNnngtc	1	4308
!	KasI Ggcgcc	2	4327	5967
!	BstXI CCANNNNNntgg	1	4415
!	NotI GCggccgc	1	4507
!	EagI Cggccg	1	4508
!	BamHI Ggatcc	1	5169
!	BspDI ATcgat	1	5476
!	NdeI CAtatg	1	5672
!	EcoRI Gaattc	1	5806
!	PsiI TTAtaa	1	6118
!	DraIII CACNNNgtg	1	6243
!	BsaAI YACgtr	1	6246
!----------------------------------------------------------------------------
	1	gacgaaaggg cCTCGTGata cgcctatttt tataggttaa tgtcatgata ataatggttt
!		BssSI.(1 2)
	61	cttaGACGTC aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
!		AatII.
	121	tctaaataca ttcaaatatG TATCCgctca tgagacaata accctgataa atgcttcaat
!		BciVI..(1 of 2)
	181	aatattgaaa aaggaagagt
!Base # 201 to 1061=ApR gene from pUC119 with some RE sites removed
!
!	1  2  3  4  5  6  7  8  9 10 11 12 13 14 15
!	fM  S  I  Q  H  F  R  V  A  L  I  P  F  F  A
	201	atg agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg
!
!		16  17  18  19  20  21  22  23  24  25  26  27  28  29  30
!		A  F  C  L  P  V  F  A  H  P  E  T  L  V  K
	246	gca ttt tgc ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa
!
!		31  32  33  34  35  36  37  38  39  40  41  42  43  44  45
!		V  K  D  A  E  D  Q  L  G  A  R  V  G  Y  I
	291	gta aaa gat gct gaa gat cag ttg ggt gcc cga gtg ggt tac atc
!
!		46  47  48  49  50  51  52  53  54  55  56  57  58  59  60
!		E  L  D  L  N  S  G  K  I  L  E  S  F  R  P
	336	gaa ctg gat ctc aac agc ggt aag atc ctt gag agt ttt cgc ccc
!
!		61  62  63  64  65  66  67  68  69  70  71  72  73  74  75
!		E  E  R  F  P  M  M  S  T  F  K  V  L  L  C
	381	gaa gaa cgt ttt cca atg atg agc act ttt aaa gtt ctg cta tgt
!
!		76  77  78  79  80  81  82  83  84  85  86  87  88  89  90
!		G  A  V  L  S  R  I  D  A  G  Q  E  Q  L  G
	426	ggc gcg gta tta tcc cgt att gac gcc ggg caa gaG CAa ctc ggT
!		BcgI............
!
!		91  92  93  94  95  96  97  98  99 100 101 102 103 104 105
!		R  R  I  H  Y  S  Q  N  D   K   V   E   Y   S   P
	471	CGc cgc ata cac tat tct cag aat gac ttg gtt gAG TAC Tca cca
!..BcgI......                                              ScaI....
!
!		106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
!		V   T   E   K   H   L   T   D   G   M   T   V   R   E   L
	516	gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa tta
!
!		121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
!		C   S   A   A   I   T   M   S   D   N   T   A   A   N   L
	561	tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta
!
!		136 137 138 139 140 141 142 143 144 145 146 147 148 149 150
!		L   L   T   I   G   G   P   K   E   L   T   A   F   L
	606	ctt ctg aca aCG ATC Gga gga ccg aag gag cta acc gct ttt ttg
!		PvuI.... (1 2)
!
!		151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
!		H   N   M   G   D   H   V   T   R   L   D   R   W   E   P
	651	cac aac atg ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg
!	166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
!		E   L   N   E   A   I   P   N   D   E   R   D   T   T   M
	696	gag ctg aat gaa gcc ata cca aac gac gag cgt gac acc acg atg
!
!		181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
!		P   V   A   M   A   T   T   L   R   K   L   L   T   G   E
	741	cct gta GCA ATG gca aca acg tTG CGC Aaa cta tta act ggc gaa
!		BsrDI..(1 2)         FspI.... (1 2)
!
!		196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
!		L   L   T   L   A   S   R   Q   Q   L   I   D   W   M   E
	786	cta ctt act cta gct tcc cgg caa caa tta ata gac tgg atg gag
!
!		211 212 213 214 215 216 217 218 219 220 221 222 223 224 225
!		A   D   K   V   A   G   P   L   L   R   S   A   L   P   A
	831	gcg gat aaa gtt gca gga cca ctt ctg cgc tcg gcc ctt ccg gct
!
!		226 227 228 229 230 231 232 233 234 235 236 237 238 239 240
!		G   W   F   I   A   D   K   S   G   A   G   E   R   G   S
	876	ggc tgg ttt att gct gat aaa tCT GGA Gcc ggt gag cgt gGG TCT
!		BpmI....(1 2)           BsaI....
!
!		241 242 243 244 245 246 247 248 249 250 251 252 253 254 255
!		R   G   I   I   A   A   L   G   P   D   G   K   P   S   R
	921	Cgc ggt atC ATT GCa gca ctg ggg cca gat ggt aag ccc tcc cgt
!BsaI......           BsrDI...(2 2)
!
!		256 257 258 259 260 261 262 263 264 265 266 267 268 269 270
!		I   V   V   I   Y   T   T   G   S   Q   A   T   M   D   E
	966	atc gta gtt atc tac acG ACg ggg aGT Cag gca act atg gat gaa
!		AhdI...........
!
!		271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
!		R   N   R   Q   I   A   E   I   G   A   S   L   I   K   H
	1011	cga aat aga cag atc gct gag ata ggt gcc tca ctg att aag cat
!
!		286 287
!		W   .
	1056	tgg taa
	1062	ctgtcagac caagtttact
	1081	catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
	1141	tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
	1201	cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
	1261	gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
	1321	taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
	1381	ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
	1441	tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
	1501	ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
	1561	cgtgcataca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
	1621	agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacagGTAT CCggtaagcg
!		BciVI.. (2 of 2)
	1681	gcagggtcgg aacaggagag cgCACGAGgg agcttccagg gggaaacgcc tggtatcttt
!		BssSI.(2 2)
	1741	atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
	1801	gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
	1861	gctggccttt tgctcACATG Ttctttcctg cgttatcccc tgattctgtg gataaccgta
!		PciI...
	1921	ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
	1981	cagtgagcga ggaagcgGAA GAGGgcccaa tacgcaaacc gcctctcccc gcgcgttggc
!		SapI....
	2041	cgattcatta atgCAGCTGg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
!		PvuII.(1 3)
	2101	acgcaatTAA TGTgagttag ctcactcatt aggcacccca ggcTTTACAc tttatgcttc
!		..-35..        Plac                  ..-10.
	2161	cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacaCAGGA AACAGCTATG
!		M13Rev_seq_primer
	2221	Accatgatta cgCCAAGCTT TGGagccttt tttttggaga ttttcaac
!		PflMI.......
	2221	ACcatgatta cgCCAAGCTT TGGagccttt tttttggaga ttttcaac
!		PflMI.......
!		Hind3.
!		signal::linker::CLight
!
!		1  2  3  4  5  6  7  8  9  10  11  12  13  14  15
!		fM   K   K   L   L   F   A   I   P   L   V   V   P   F   Y
	2269	gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat
!
!		Linker.............................. End of FR4
!		16  17  18  19  20  21  22  23  24  25  26  27  28  29  30
!		S   H   S   A   Q   V   Q   L   Q   V   D   L   E   I   K
	2314	tct cac aGT GCA Cag gtc caa CTG CAG GTC GAC CTC GAG atc aaa
!		ApaLI......          PstI...        XhoI...
!		BspMI...
!		SalI...
!		AccI...(1 2)
!		HincII.(1 2)
!
!		Vlight domains could be cloned in as ApaLI-XhoI fragments.
!		VL-CL (kappa) segments can be cloned in as ApaLI-AscI fragments.
!
!		Ckappa----------------------------------------------------
!		31  32  33  34  35  36  37  38  39  40  41  42  43  44  45
!		R   G   T   V   A   A   P   S   V   F   I   F   P   P   S
	2359	cgt gga act gtg gct gca cca tct GTC TTC atc ttc ccg cca tct
!		BbsI...(1 2)
!
!		46  47  48  49  50  51  52  53  54  55  56  57  58 59  60
!		D   E   Q   L   K   S   G   T   A   S   V   V   C   L   L
	2404	gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg
!
!		61  62  63  64  65  66  67  68  69  70  71  72  73  74  75
		N   N   F   Y   P   R   E   A   K   V   Q   W   K   V   D
	2449	aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat
!
!		76  77  78  79  80  81  82  83  84  85  86  87  88  89  90
!		N   A   L   Q   S   G   N   S   Q   E   S   V   T   E   Q
	2494	aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag
!
!		91  92  93  94  95  96  97  98  99 100 101 102 103 104 105
!		D   S   K   D   S   T   Y   S   L   S   S   T   L   T   L
	2539	gac agc aag gac agc acc tac agc ctc agc agc acc ctg acG CTG
!		EspI...
!
!		106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
!		S   K   A   D   Y   E   K   H   K   V   Y   A   C   E   V
	2584	AGC aaa gca gac tac gag aaa cac aaa GTC TAC gcc tgc gaa gtc
!		...EspI....	AccI...(2 2)
!		121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
!		T   H   Q   G   L   S   S   P   V   T   K   S   F   N   R
	2629	acc cat cag ggc ctg agt tcA CCG GTg aca aag agc ttc aac agg
!		AgeI....(1 2)
!
!		136 137 138 139 140
!		G   E   C   .   .
	2674	gga gag tgt taa taa GG CGCGCCaatt
!		AscI.....
!		BssHII.
	2701	ctatttcaag gagacagtca ta
!
!		PelB::3-23(stuffed)::CHl::III fusion gene
!
!		1   2   3   4   5   6   7   8   9  10  11  12  13  14  15
!		M   K   Y   L   L   P   T   A   A   A   G   L   L   L   L
	2723	atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc
!
!		----------------------------------------
!
!		16  17  18  19  20  21  22
!		A   A   Q   P   A   M   A
	2768	gcG GCC cag ccG GCC atg gcc
!		SfiI..............
!		NgcMIV..(1 2)
!		NcoI....
!
!		FRl(DP47 V3-23)-------------------
!		23  23  24  25  26  27  28  29  30
!		E   V   Q   L   L   E   S   G
	2789	gaa|gtt|CAA|TTG|tta|gag|tct|ggt|
!		|MfeI|
!
!		-------------FRl---------------------------------------------
!		31  32  33  34  35  36  37  38  39  40  41  42  43  44  45
!		G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A
	2813	|ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct|
!
!		----FRl-----
!		46  47  48
!		A   S   G
	2858	|gct|TCC|GGA|
!		|BspEI|
!
!		Stuffer for CDR1, FR2, and CDR2----------------------------------
!		There are no stp codons in this stuffer.
	2867	gcttcAGATC Tgtttgcctt
!		BglII..
	2887	tttgtggggt ggtgcagatc gcgttacgga gatcgaccga ctgcttgagc aaaagccacg
	2947	cttaactgcT GATCAggcat gggatgttat tcgccaaacc agtcgtcagg atcttaacct
!		BclT...
	3007	gaggcttttt ttacctactc tgcaagcagc gacatctggt ttgacacaga gcgatccgcg
	3067	tcgtcagttg gtagaaacat taacacgttg ggatggcatc aatttgctta atgatgatgg
	3127	taaaacctgg cagcagccag gctctgccat cctgaacgtt tggctgacca gtatgttgaa
	3187	gcgtaccgta gtggctgccg tacctatgCC Atttgataag TGGtacagcg ccagtggcta
!		XcmI..............
	3247	cgaaacaacc caggacggcc caactggttc gctgaatata agtgttggag caaaaatttt
	3307	gtatgaggcg gtgcagggag acaaatcacc aatcccacag gcggttgatc tgtttgctgg
	3367	gaaaccacag caggaggttg tgttggctgc gctggaagat acctgggaga ctctttccaa
	3427	acgctatggc aataatgtga gtaactggaa aacacctgca atggccttaa cgttccgggc
	3487	aaataatttc tttggtgtac cgcaggccgc agcggaagaa ACGCGTcatc aggcggagta
!		MluI..
	3547	tcaaaaccgt ggaacagaaa acgatatgat tgttttctca ccaacgacaa gcgatcgtcc
	3607	tgtgcttgcc tgggatgtgg tcgcacccgg tcagagtggg tttattgctc ccgatggaac
	3667	agttgataag cactatgaag atcagctgaa aatgtacgaa aattttggcc gtaagtcgct
!		PvuII.
	3727	ctgGTTAACg aagcaggatg tggaggcgca taaggagtcg
!		HpaI..
!		HincII(2 2)
!
!		-------FR3------------------------------------------------
!		4   5   6   7   8   9  10  11  12  13  14  15  16
!		93  94  95  96  97  98 99 100 101 102 103 104 105
!		S   R   D   N   S   K   N  T   L   Y   L   Q   M
	3767	|TCT|AGA|aga|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg
!		|XbaI|
!
!		---FR3----------------------------------------------------|
!		17  18  19  20
!		106 107 108 109
!		N   S   L   s   l   s   i   t   s   q
	3806	|aac|agC|TTA|Ag t ctg agc att CGG TCC G
!		|AflII|               RsrII..
!
!		g   h   s   p   t   .
	3834	gg caa cat tct cca aac tga ccagacga cacaaacggc
	3872	ttacgctaaa tcccgcgcat gggatggtaa agaggtggcg tctttgctgg cctggactca
	3934	tcagatgaag gccaaaaatt ggcaggagtg gacacagcag gcagcgaaac aagcactgac
	3992	catcaactgg tactatgctg atgtaaacgg caatattggt tatgttcata ctggtgctta
	4052	tccagatcgt caatcaggcc atgatccgcg attacccgtt cctggtacgg gaaaatggga
	4112	ctggaaaggg ctattgcctt ttgaaatgaa ccctaaggtg tataaccccc ag
	4164	aa GCTAGC ctgcggcttc
!		NheI..
!
	4182	G|GTC|ACC|                                       gtc tca agc
!		|BstEII|
!
!		137 137 138 139 140 141 142 143 144 145 146 147 148 149 150
!		A   S   T   K   G   P   S   V   F   P   L   A   P   S   S
	4198	gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc
!
!		151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
!		K   S   T   S   G   G   T   A   A   L   G   C   L   V   K
	4243	aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag
!
!		166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
!		D   Y   F   P   E   P   V   T   V   S   W   N   S   G   A
	4288	gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc
!
!		181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
!		L   T   S   G   V   H   T   F   P   A   V   L   Q   S   S
	4333	ctg acc agc ggc gtc cac acc ttc ccg gct gtc cta cag tcc tca
!
!		196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
!		G   L   Y   S   L   S   S   V   V   T   V   P   S   S   S
	4378	gga ctc tac tcc ctc agc agc gta gtg acc gtg ccc tcc agt agc
!
!		211 212 213 214 215 216 217 218 219 220 221 222 223 224 225
!		L   G   T   Q   T   Y   I   C   N   V   N   H   K   P   S
	4423	ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc
!
!		226 227 228 229 230 231 232 233 234 235 236 237 238 239 240
!		N   T   K   V   D   K   K   V   E   P   K   S   C
	4468	aac acc aag gtg gac aaG AAA GTT GAG CCC AAA TCT TGT
!		CN-TQHCforw......................
!
!		Poly His linker
!		139 140 141 142 143 144 145 146 147 148 149 150
!		A   A   A   H   H   H   H   H   H   G   A   A
	4507	GCG GCC GCa cat cat cat cac cat cac ggg gcc gca
!		NotI......
!		EagI....
!
!		151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
!		E   Q   K   L   I   S   E   E   D   L   N   G   A   A
	4543	gaa caa aaa ctc atc tca gaa gag gat ctg aat ggg gcc gca tag
!
!		Mature III--------------------------------------------------...
!		166 167 168 169 170 171 172 173 174 175 176 177 178 178 180
!		T   V   E   S   C   L   A   K   P   H   T   E   N   S   F
	4588	act ggt gaa agt tgt tta gca aaa cct cat aca gaa aat tca ttt
!
!		181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
!		T   N   V   W   K   D   D   K   T   L   D   R   Y   A   N
	4633	act aac gtc tgg aaa gac gac aaa act tta gat cgt tac gct aac
!
!		196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
!		Y   E   G   C   L   W   N   A   T   G   V   V   V   C   T
	4678	tat gag ggc tgt ctg tgG AAT GCt aca ggc gtt gtg gtt tgt act
!		BsmI....
!
!		211 212 213 214 215 216 217 218 219 220 221 222 223 224 225
!		G   D   E   T   Q   C   Y   G   T   W   V   P   I   G   L
	4723	ggt gac gaa act cag tgt tac ggt aca tgg gtt cct att ggg ctt
!
!		226 227 228 229 230 231 232 233 234 235 236 237 238 239 240
!		A   I   P   E   N   E   G   G   G   S   E   G   G   G   S
	4768	gct atc cct gaa aat gag ggt ggt ggc tct gag ggt ggc ggt tct
!
!		241 242 243 244 245 246 247 248 249 250 251 252 253 254 255
!		E   G   G   G   S   E   G   G   G   T   K   P   P   E   Y
	4813	gag ggt ggc ggt tct gag ggt ggc ggt act aaa cct cct gag tac
!
!		256 257 258 259 260 261 262 263 264 265 266 267 268 269 270
!		G   D   T   P   I   P   G   Y   T   Y   I   N   P   L   D
	4858	ggt gat aca cct att ccg ggc tat act tat atc aac cct ctc gac
!
!		271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
!		G   T   Y   P   P   G   T   E   Q   N   P   A   N   P   N
	4903	ggc act tat ccg cct ggt act gag caa aac ccc gct aat cct aat
!
!		286 287 288 289 290 291 292 293 294 295 296 297 298 299 300
!		P   S   L   E   E   S   Q   P   L   N   T   F   M   F   Q
	4948	cct tct ctt GAG GAG tct cag cct ctt aat act ttc atg ttt cag
!		BseRI..(2 2)
!
!		301 302 303 304 305 306 307 308 309 310 311 312 313 314 315
!		N   N   R   F   R   N   R   Q   G   A   L   T   V   Y   T
	4993	aat aat agg ttc cga aat agg cag ggt gca tta act gtt tat acg
!
!		316 317 318 319 320 321 322 323 324 325 326 327 328 329 330
!		G   T   V   T   Q   G   T   D   P   V   K   T   Y   Y   Q
	5038	ggc act gtt act caa ggc act gac ccc gtt aaa act tat tac cag
!
!		331 332 333 334 335 336 337 338 339 340 341 342 343 344 345
!		Y   T   P   V   S   S   K   A   M   Y   D   A   Y   W   N
	5083	tac act cct gta tca tca aaa gcc atg tat gac gct tac tgg aac
!
!		346 347 348 349 350 351 352 353 354 355 356 357 358 359 360
!		G   K   P   R   D   C   A   F   H   S   G   P   N   E   D
	5128	ggt aaa ttc aga gac tgc gct ttc cat tct ggc ttt aat gaG GAT
!		BamHI..
!
!		361 362 363 364 365 366 367 368 369 370 371 372 373 374 375
!		P   F   V   C   E   Y   Q   G   Q   S   S   D   L   P   Q
	5173	CCa ttc gtt tgt gaa tat caa ggc caa tcg tct gAC CTG Cct caa
!	BAMHI...                                  BspMI...(2 2)
!
!		376 377 378 379 380 381 382 383 384 385 386 387 388 389 390
!		P   P   V   N   A   G   G   G   S   G   G   G   S   G   G
	5218	cct cct gtc aat gct ggc ggc ggc tct ggt ggt ggt tct ggt ggc
!
!		391 392 393 394 395 396 397 398 399 400 401 402 403 404 405
!		G   S   E   G   G   G   S   E   G   G   G   S   E   G   G
	5263	ggc tct gag ggt ggc ggc tct gag ggt ggc ggt tct gag ggt ggc
!
!		406 407 408 409 410 411 412 413 414 415 416 417 418 419 420
!		G   S   E   G   G   G   S   G   G   G   S   G   S   G   D
	5308	ggc tct gag ggt ggc ggt tcc ggt ggc ggc tcc ggt tcc ggt gat
!
!		421 422 423 424 425 426 427 428 429 430 431 432 433 434 435
!		F   D   Y   E   K   M   A   N   A   N   K   G    A   M   T
	5353	ttt gat tat gaa aaa atg gca aac gct aat aag ggg gct atg acc
!
!		436 437 438 439 440 441 442 443 444 445 446 447 448 449 450
!		E   N   A   D   E   N   A   L   Q   S   D   A   K   G   K
	5398	gaa aat gcc gat gaa aac gcg cta cag tct gac gct aaa ggc aaa
!
!		451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
!		L   D   S   V   A   T   D   Y   G   A   A   I   D   G   F
	5443	ctt gat tct gtc gct act gat tac ggt gct gct ATC GAT ggt ttc
!		BspDI..
!
!		466 467 468 469 470 471 472 473 474 475 476 477 478 479 480
!		I   G   D   V   S   G   L   A   N   G   N   G   A   T   G
	5488	att ggt gac gtt tcc ggc ctt gct aat ggt aat ggt gct act ggt
!
!		481 482 483 484 485 486 487 488 489 490 491 492 493 494 495
!		D   F   A   G   S   N   S   Q   M   A   Q   V   G   D   G
	5533	gat ttt gct ggc tct aat tcc caa atg gct caa gtc ggt gac ggt
!
!		496 497 498 499 500 501 502 503 504 505 506 507 508 509 510
!		D   N   S   P   L   M   N   N   F   R   Q   Y   L   P   S
	5578	gat aat tca cct tta atg aat aat ttc cgt caa tat tta cct tct
!
!		511 512 513 514 515 516 517 518 519 520 521 522 523 524 525
!		L   P   Q   S   V   E   C   R   P   Y   V   F   G   A   G
	5623	ttg cct cag tcg gtt gaa tgt cgc cct tat gtc ttt ggc gct ggt
!
!		526 527 528 529 530 531 532 533 534 535 536 537 538 539 540
!		K   P   Y   E   P   S   I   D   C   D   K   I   N   L   F
	5668	aaa cCA TAT Gaa ttt tct att gat tgt gac aaa ata aac tta ttc
!		NdeI
!
!		541 542 543 544 545 546 547 548 549 550 551 552 553 554 555
!		R   G   V   F   A   F   L   L   Y   V   A   T   F   M   Y
	5713	cgt ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat
!
!		556 557 558 559 560 561 562 563 564 565 566 567 568 569 570
!		V   F   S   T   F   A   N   I   L   R   N   K   E   S   .
	5758	gta ttt tcg acg ttt gct aac ata ctg cgt aat aag gag tct taa
!
!	571
!		.
	5803	taa GAATTC
!		EcoRI.
	5812	actggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
	5871	gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcacCGATC
!
	5931	Gcccttccca acagtTGCGC Agcctgaatg gcgaatGGCG CCtgatgcgg tattttctcc
!...PvuI... (3 3)                FspI... (2 2)          KasI...(2 2)
	5991	ttacgcatct gtgcggtatt tcacaccgca tataaattgt aaacgttaat attttgttaa
	6051	aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
	6111	aaatcccTTA TAAatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga
!		PsiI...
	6171	acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
	6231	agggcgatgg ccCACtacGT Gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc
!		DraIII....
	6291	gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaaGC
!		NgoMIV..
	6351	CGGCgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
!..NGoMIV.(2 2)
	6411	caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
	6471	agggcgcgta ctatggttgc tttgacgggt gcagtctcag tacaatctgc tctgatgccg
	6531	catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
	6591	tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
	6651	ggttttcacc gtcatcaccg aaacgcgcga

[0179]

TABLE 630
Oligonucleotides used to clone CDR1/2 diversity
All sequences are 5′ to 3′.
1) on_CD1Bsp, 30 bases
A c c T c A c T g g c T T c c g g A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
T T c A c T T T c T c T
19 20 21 22 23 24 25 26 27 28 29 30
2) ON_Br12, 42 bases
A g A A A c c c A c T c c A A A c c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
T T T A c c A g g A g c T T g g c g
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
A A c c c A
37 38 39 40 41 42
3) ON_CD2Xba, 51 bases
g g A A g g c A g T g A T c T A g A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
g A T A g T g A A g c g A c c T T T
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
A A c g g A g T c A g c A T A
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
4) ON_BotXba, 23 bases
g g A A g g c A g T g A T c T A g A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
g A T A g
19 20 21 22 23

Claims

1. A method for cleaving single-stranded nucleic acid sequences at a desired location, the method comprising the steps of: (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

2. A method for cleaving single-stranded nucleic acid sequences at a desired location, the method comprising the steps of: (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type II-S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site; and (ii) cleaving the nucleic acid solely at the Type 11-S cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide; the contacting and the cleaving steps being performed a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

3. In a method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a part of the diversity of the family, the improvement being characterized in that the displayed at least a part of peptide, polypeptide or protein is encoded at least in part by a nucleic acid that has been cleaved at a desired location by a method comprising the steps of: (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

4. In a method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a part of the diversity of the family, the improvement being characterized in that the displayed peptide, polypeptide or protein is encoded by a DNA sequence comprising a nucleic acid that has been cleaved at a desired location by (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type II-S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site; and (ii) cleaving the nucleic acid solely at the Type II-S cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desires location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

5. A method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a part of the diversity of the family, the method comprising the steps of: (i) preparing a collection of nucleic acids that code at least in part for members of the diverse family; (ii) rendering the nucleic acids single-stranded; (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of: (a) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (b) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;  the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature; and (iv) displaying a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids on the surface of the genetic package and collectively displaying at least a portion of the diversity of the family.

6. A method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a portion of the diversity of the family, the method comprising the steps of: (i) preparing a collection of nucleic acids that code, at least in part, for members of the diverse family; (ii) rendering the nucleic acids single-stranded; (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of: (a) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type II-S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site; and (b) cleaving the nucleic acid solely at the Type II-S cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;  the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a cleavage endonuclease that is active at the chosen temperature; and (iv) displaying a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids on the surface of the genetic package and collectively displaying at least a portion of the diversity of the family.

7. A library comprising a collection of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and collectively display at least a portion of the diversity of the family, the library being produced using the methods of claims 3, 4, 5 or 6.

8. A library comprising a collection of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and that collectively display at least a portion of the family, the displayed peptides, polypeptides or proteins being encoded by DNA sequences comprising at least in part sequences produced by cleaving single-stranded nucleic acid sequences at a desired location by a method comprising the steps of: (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

9. A library comprising a collection of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and that collectively display at least a portion of the diversity of the family of the displayed peptides, polypeptides or proteins being encoded by DNA sequences comprising at least in part sequences produced by cleaving single-stranded nucleic acid sequences at a desired location by a method comprising the steps of: (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a Type II S restriction endonuclease recognition site, whose cleavage site is located at a known distance from the recognition site where the cleavage of the nucleic acid is desired; and (ii) cleaving the nucleic acid solely at the Type II-S cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

10. The methods according to any one of claims 1 to 9, wherein the nucleic acids encode at least a portion of an immunoglobulin.

11. The methods according to claim 10, wherein the immunoglobulin comprises a Fab or single chain Fv.

12. The methods according to claim 10 or 11, wherein the immunoglobin comprises at least portion of a heavy chain.

13. The methods according to claim 12, wherein at least a portion of the heavy chain is human.

14. The methods according to claim 10 or 11, wherein the immunoglobulin comprises at least a portion of FR1.

15. The methods according to claim 14, wherein at least a portion of the FR1 is human.

16. The methods according to claim 10 or 11, wherein the immunoglobulin comprises at least a portion of a light chain.

17. The methods according to claim 16, wherein at least a portion of the light chain is human.

18. The methods according to any one of claims 1 to 9, wherein the nucleic acid sequences are at least in part derived from patients suffering from at least one autoimmune disease and/or cancer.

19. The methods according to claim 18, wherein the autoimmune disease is selected from the group comprising lupus, erythematosus, systemic sclerosis, rheumatoid arthritis, antiphosolipid syndrome or vasculitis.

20. The methods according to claim 18, wherein the nucleic acids are at least in part isolated from the group comprising peripheral blood cells, bone marrow cells spleen cells or lymph node cells.

21. The methods according to claim 5 or 6 further comprising an nucleic acid amplification step between steps (i) and (ii), between steps (ii) and (iii) or between steps (iii) and (iv).

22. The methods according to claim 21, wherein the amplification step uses geneRACE™.

23. The methods according to any one of claims 1 to 9, wherein the temperature is between 45° C. and 75° C.

24. The methods according to claim 23, wherein the temperature is between 50° C. and 60° C.

25. The methods according to claim 24, wherein the temperature is between 55° C. and 60° C.

26. The methods according to claim 1, 3, 5 or 8, wherein the length of the single-stranded oligonucleotide is between 17 and 30 bases.

27. The methods according to claim 26, wherein the length of the single-stranded oligonucleotide is between 18 and 24 bases.

28. The methods according to claim 1, 3, 5 or 8, wherein the restriction endonuclease is selected from the group comprising MaelII, Tsp45I, HphI, BsaJI, AluI, BlpI, DdeI, BglII, MslI, BsiEI, EaeI, EagI, HaeIII, Bst4CI, HpyCH41II, HinfI, MlyI, PleI, MnlI, HpyCH4V, BsmAI, BpmI, XmnI, or SacI.

29. The methods according to claim 28, wherein the restriction endonuclease is selected from the group comprising Bst4CI, TaaI, HpyCH4III, BlpI, HpyCH4V or MslI.

30. The methods according to claim 2, 4, 6 or 9, wherein the length of the single-stranded region of the partially double-stranded oligonucleotide is between 14 and 22 bases.

31. The methods according to claim 30, wherein the length of the single-stranded region of the partially double-stranded oligonucleotide is between 14 and 17 bases.

32. The methods according to claim 31, wherein the length of the single-stranded region of the oligonucleotide is between 18 and 20 bases.

33. The methods according to claim 2, 4, 6 or 9, wherein the length of the double-stranded region of the partially double-stranded oligonucleotide is between 10 and 14 base pairs formed by a stem and its palindrome.

34. The methods according to claim 33 wherein, the partially double-stranded oligonucleotide comprises a loop of 3 to 8 bases between the stem and the palindrome.

35. The methods according to claim 2, 4, 6 or 9, wherein the Type II-S restriction endonuclease is selected from the group comprising AarICAC, AceIII, Bbr7I, BbvI, BbvII, Bce83I, BceAI, BcefI, BciVI, BfiI, BinI, BscAI, BseRI, BsmFI, BspMI, EciI, Eco57I, FauI, FokI, GsuI, HgaI, HphI, MboII, MlyI, MmeI, MnlI, PleI, RleAI, SfaNI, SspD5I, Sthl32I, StsI, TaqII, Tth111II, or UbaPI.

36. The methods according to claim 35, wherein the Type II-S restriction endonuclease is FokI.

37. A method for preparing single-stranded nucleic acids for cloning into an vector, the method comprising the steps of: (i) contacting a single-stranded nucleic acid sequence that has been cleaved with a restriction endonuclease with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region that remains after cleavage, the double-stranded region of the oligonucleotide including any sequences necessary to return the sequences that remain after cleavage into proper and original reading frame for expression and containing a restriction endonuclease recognition site 5′ of those sequences; and (ii) cleaving the partially double-stranded oligonucleotide sequence solely at the restriction endonuclease recognition site contained within the double-stranded region of the partially double-stranded oligonucleotide.

38. The method according to claim 37, wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 2 and 15 bases.

39. The method according to claim 38, wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 7 and 10 bases.

40. The method according to claim 37, wherein the length of the double-stranded portion of the partially double-stranded oligonucleotide is between 12 and 100 base pairs.

41. The method according to claim 40, wherein the length of the double-stranded portion of the partially double-stranded oligonucleotide is between 20 and 100 base pairs.