Vectors for directional cloning

Abstract

The invention provides vectors and methods for directional cloning.

1 . A method for the directional subcloning of DNA fragments comprising: 
 a) providing a first vector comprising a first selectable marker gene and a DNA sequence of interest, which DNA sequence of interest is flanked by at least two restriction enzyme sites, wherein at least one of the flanking restriction enzyme sites is a site for a first restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates complementary single-strand DNA overhangs, wherein at least one of the flanking restriction enzyme sites is for a second restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates ends that are not complementary to the overhangs generated by the first restriction enzyme, wherein digestion of the first vector with the first restriction enzyme and the second restriction enzyme site generates a first linear DNA fragment which lacks the first selectable marker gene but comprises the DNA sequence of interest;    b) providing a second vector comprising a second selectable marker gene which is distinguishable from the first selectable marker gene and non-essential DNA sequences, optionally including a counterselectable gene, which non-essential sequences are flanked by at least two restriction enzymes sites, wherein at least one of the flanking restriction enzyme sites in the second vector is for a third restriction enzyme which generates complementary single-strand DNA overhangs that are complementary to the single-strand DNA overhang generated by the first restriction enzyme in the first linear DNA fragment, wherein at least one of the flanking restriction sites in the second vector is for a fourth restriction enzyme which generates ends that are not complementary to the ends generated by the first or third restriction enzyme but can be ligated to the ends generated by the second restriction enzyme, and wherein digestion of the second vector with the third restriction enzyme and the fourth restriction enzyme generates a second linear DNA fragment which lacks non-essential DNA sequences but comprises the second selectable marker, which second linear DNA fragment is flanked by ends which permit the oriented joining of the first linear DNA fragment to the second linear DNA fragment; and    c) combining the first and second vectors, the first vector and the second linear DNA fragment, or the second vector and the first linear DNA fragment in a suitable buffer with one or more restriction enzymes and optionally DNA ligase under conditions effective to result in digestion and optionally ligation to yield a mixture optionally comprising a third vector comprising the first and second linear DNA molecules which are joined in an oriented manner.    
     
     
         2 . The method of  claim 1  wherein the second restriction enzyme generates blunt ends and the first linear DNA fragment is flanked by a first single-strand DNA overhang and a blunt end.  
     
     
         3 . The method of  claim 1  wherein the first and third restriction enzymes are not the same.  
     
     
         4 . The method of  claim 1  wherein the second and fourth restriction enzymes are not the same.  
     
     
         5 . The method of  claim 1  wherein the second and fourth restriction enzymes generate blunt ends.  
     
     
         6 . The method of  claim 1  wherein the first restriction enzyme is SgfI.  
     
     
         7 . The method of  claim 6  wherein the second restriction enzyme is PmeI.  
     
     
         8 . The method of  claim 1  wherein the third restriction enzyme generates a 3′ TA overhang.  
     
     
         9 . The method of  claim 8  wherein the third restriction enzyme is PvuI or PacI.  
     
     
         10 . The method of  claim 1  wherein the DNA sequence of interest comprises an open reading frame comprising one or more sites for the first or second restriction enzyme.  
     
     
         11 . The method of  claim 10  wherein prior to digestion with the one or more restriction enzymes, the sites for the one or more restriction enzymes in the open reading frame are protected so as to prevent digestion.  
     
     
         12 . The method of  claim 11  wherein the sites are protected by methylation.  
     
     
         13 . The method of  claim 12  wherein prior to methylation the flanking sites for the first or second restriction enzyme are contacted with an oligonucleotide complementary to the flanking restriction enzyme site and RecA.  
     
     
         14 . A vector system for cloning comprising: 
 a first vector comprising a first selectable marker gene and a DNA sequence of interest, which DNA sequence of interest is flanked by at least two restriction enzyme sites, wherein at least one of the flanking restriction enzyme sites is a site for a first restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates complementary single-strand DNA overhangs, wherein at least one of the flanking restriction enzyme sites is for a second restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates ends that are not complementary to the overhangs generated by the first restriction enzyme, wherein digestion of the first vector generates a first linear DNA fragment which lacks the first selectable marker gene but comprises the DNA sequence of interest, wherein the restriction enzyme sites are designed such that the first linear DNA fragment can be religated directly to a second vector comprising a second selectable marker gene which is distinguishable from the first selectable marker gene and non-essential DNA sequences, optionally including a counterselectable gene, which non-essential DNA sequences are flanked by at least two restriction enzymes sites, wherein at least one of the flanking restriction enzyme sites in the second vector is for a third restriction enzyme which generates complementary single-strand DNA overhangs which are complementary to the single-strand DNA overhangs generated by the first restriction enzyme, wherein at least one of the flanking restriction sites in the second vector is for a fourth restriction enzyme which generates ends that are not complementary to the ends generated by the first or third restriction enzyme but can be ligated to the ends generated by the second restriction enzyme, wherein digestion of the second vector with the third and fourth restriction enzymes generates a second linear DNA fragment which lacks the non-essential DNA sequences but comprises the second selectable marker gene, wherein the second linear DNA fragment is flanked by ends which permit the oriented joining of the first linear DNA fragment to the second linear DNA fragment.    
     
     
         15 . The vector system of  claim 14  wherein the second restriction enzyme generates blunt ends and the first linear DNA fragment is flanked by a first single-strand DNA overhang and a blunt end.  
     
     
         16 . The vector system of  claim 14  wherein the first and third restriction enzymes are not the same.  
     
     
         17 . The vector system of  claim 14  wherein the second and fourth restriction enzymes are not the same.  
     
     
         18 . The vector system of  claim 14  wherein the second and fourth restriction enzymes generate blunt ends.  
     
     
         19 . The vector system of  claim 14  wherein ligation and oriented jointing yields a third vector encoding a N-terminal fusion protein which is encoded by the DNA sequence of interest and nucleic acid sequences 5′ to the 3′ end of the second linear DNA fragment.  
     
     
         20 . The vector system of  claim 14  wherein ligation and oriented joining yields a third vector encoding a C-terminal fusion protein which is encoded by the DNA sequence of interest and nucleic acid sequences 3′ to the 5′ end of the second linear DNA fragment.  
     
     
         21 . The vector system of  claim 14  wherein ligation and oriented joining yields a third vector encoding a fusion protein which is encoded by the DNA sequence of interest and nucleic acid sequences 5′ and 3′ to the respective 3′ and 5′ end of the second linear DNA fragment.  
     
     
         22 . The vector system of  claim 14  wherein ligation and oriented joining yields a third vector encoding a fusion protein encoded by the DNA sequence of interest and the exchange site(s) created by the oriented joining.  
     
     
         23 . The vector system of  claim 14  wherein one of the restriction enzymes is AarI, AscI, BbrCI, CspI, DraI, FseI, NotI, NruI, PacI, PmeI, PvuI, SapI, SdaI, SfiI, SgfI, SplI, SrfI, SwaI, or a restriction enzyme which has the same recognition site as AarI, AscI, BbrCI, CspI, DraI, FseI, NotI, NruI, PacI, PmeI, PvuI, SapI, SdaI, SfiI, SgfI, SplI, SrfI, SwaI.  
     
     
         24 . The vector system of  claim 20 ,  21  or  22  wherein the fusion protein is a GST fusion protein, GFP fusion protein, thioredoxin fusion protein, maltose binding protein fusion protein, protease cleavage site fusion protein, metal binding domain fusion protein or dehalogenase fusion protein.  
     
     
         25 . The vector system of  claim 20 ,  21  or  22  wherein the fusion protein is more soluble, easier to purify or easier to detect relative to the corresponding non-fusion protein.  
     
     
         26 . A kit comprising the vector system of  claim 14 .  
     
     
         27 . A method for producing a vector suitable for expression of an amino acid sequence of interest, comprising: 
 combining at least two vectors in a suitable buffer with one or more restriction enzymes and optionally DNA ligase under conditions effective to result in digestion and optionally ligation to yield a mixture optionally comprising a third vector, wherein a first vector comprises a first selectable marker gene and a DNA sequence of interest, which DNA sequence of interest is flanked by at least two restriction enzyme sites, wherein two or more of the flanking restriction enzyme sites are sites for a first restriction enzyme which is a hapaxoterministic restriction enzyme, wherein digestion of the first vector with the first restriction enzyme generates a first linear DNA fragment which lacks the first selectable marker gene but comprises the DNA sequence of interest and a first pair non-self complementary single-strand DNA overhangs, wherein a second vector comprises a second selectable marker gene which is distinguishable from the first selectable marker gene and non-essential DNA sequences that optionally include a counterselectable gene, which non-essential DNA sequences are flanked by two or more restriction enzyme sites, wherein two or more of the flanking sites in the second vector are for a second restriction enzyme which is a hapaxoterministic restriction enzyme, wherein digestion of the second vector with the second restriction enzyme generates a second linear DNA fragment which lacks non-essential DNA sequences but comprises the second selectable marker gene and a second pair of non-self complementary single-strand DNA overhangs, wherein each of the second pair of the non-self-complementary DNA overhangs is complementary to only one of the single-strand DNA overhangs of the first pair of non-self complementary single-strand DNA overhangs and permits the oriented joining of the first linear DNA fragment to the second linear DNA fragment.    
     
     
         28 . A method for producing a vector suitable for expression of an amino acid sequence of interest, comprising: 
 combining at least two vectors in a suitable buffer with one or more restriction enzymes and optionally DNA ligase under conditions effective to result in digestion and optionally ligation to yield a mixture optionally comprising a third vector, wherein a first vector comprises a first selectable marker gene and a DNA sequence of interest, which DNA sequence of interest is flanked by at least two restriction enzyme sites, wherein at least one of the flanking restriction enzyme sites is a site for a first restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates complementary single-strand DNA overhangs, wherein at least one of the flanking restriction enzyme sites is for a second restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates ends that are not complementary to the overhangs generated by the first restriction enzyme, wherein digestion of the first vector generates a first linear DNA fragment which lacks the first selectable marker gene but comprises the DNA sequence of interest, wherein a second vector comprises a second selectable marker gene which is distinguishable from the first selectable marker gene and non-essential DNA sequences, optionally including a counterselectable gene, which non-essential DNA sequences are flanked by at least two restriction enzymes sites, wherein at least one of the flanking restriction enzyme sites in the second vector is for a third restriction enzyme which generates single-strand DNA overhangs which are complementary to the single-strand DNA overhangs generated by the first restriction enzyme, wherein at least one of the flanking restriction sites in the second vector is for a fourth restriction enzyme that which generates ends that are not complementary to the ends generated by the first or third restriction enzyme but can be ligated to the ends generated by the second restriction enzyme, and wherein digestion of the second vector with the third and fourth restriction enzymes generates a second linear DNA fragment which lacks the non-essential DNA sequences but comprises the second selectable marker gene, wherein the second linear DNA fragment is flanked by ends which permit the oriented joining of the first linear DNA fragment to the second linear DNA fragment.    
     
     
         29 . The method of  claim 28  wherein the second restriction enzyme generates blunt ends and the first linear DNA fragment is flanked by a first single-strand DNA overhang and a blunt end.  
     
     
         30 . The method of  claim 28  wherein the first and third restriction enzymes are not the same.  
     
     
         31 . The method of  claim 28  wherein the second and fourth restriction enzymes are not the same.  
     
     
         32 . The method of  claim 28  wherein the second and fourth restriction enzymes generate blunt ends.  
     
     
         33 . The method of  claim 28  wherein one of the restriction enzymes is a class IIS restriction enzyme.  
     
     
         34 . The method of  claim 33  wherein the class IIS restriction enzyme is AccB7I, AceIII, AclWI, AdeI, AhdI, Alw26I, AlwI, AlwNI, ApaBI, AspEI, AspI, AsuHPI, BbsI, BbvI, BbvII, Bce83I, BcefI, BciVI, BfiI, BglI, BinI, BmrI, BpiI, BpmI, BpuAI, BsaI, Bse3DI, Bse4I, BseGI, BseLI, BseRI, BsgI, BslI, BsmAI, BsmBI, BsmFI, BspMI, BsrDI, Bst71I, BstAPI, BstF5I, BstXI, Bsu6I, DraIII, DrdI, DseDI, Eam1104I, Eam1105I, EarI, EchHKI, Eco31I, Eco57I, EcoNI, i1396I, Esp3I, FokI, FauI, GsuI, HgaI, HphI, MboII, MsiYI, MwoI, NruGI, PflMI, PflFI, PleI, SfaNI, TspRI, Ksp632I, MmeI, RleAI, SapI, SfiI, TaqII, Tth111I, Tth111II, Van91I, XagI, XcmI, or a restriction enzyme which has the same recognition site as AccB7I, AceIII, AclWI, AdeI, AhdI, Alw26I, AlwI, AlwNI, ApaBI, AspEI, AspI, AsuHPI, BbsI, BbvI, BbvII, Bce83I, BcefI, BciVI, BfiI, BglI, BinI, BmrI, BpiI, BpmI, BpuAI, BsaI, Bse3DI, Bse4I, BseGI, BseLI, BseRI, BsgI, BslI, BsmAI, BsmBI, BsmFI, BspMI, BsrDI, Bst71I, BstAPI, BstF5I, BstXI, Bsu6I, DraIII, DrdI, DseDI, EamI 104I, Eam1105I, EarI, EchHKI, Eco31I, Eco57I, EcoNI, i1396I, Esp3I, FokI, FauI, GsuI, HgaI, HphI, MboII, MsiYI, MwoI, NruGI, PflMI, PflFI, PleI, SfaNI, TspRI, Ksp632I, MmeI, RleAI, SapI, SfiI, TaqII, Tth111I, Tth111II, Van91I, XagI, XcmI.  
     
     
         35 . The method of  claim 33  wherein one of the restriction enzymes is AvaI, Ama87I, BcoI, BsoBI, Eco88I, AvaII, Eco47I, Bme18I, HgiEI, SinI, BanI, AccB1I, BshNI, Eco64I, BfmI, BstSFI, SfcI, Bpu10I, BsaMI, BscCI, BsmI, Mva1269I, Bsh1285I, BsaOI, BsiEI, BstMCI, BselI, BseNI, BsrI, Cfr10I, BsiI, BssSI, Bst2BI, BsiZI, AspS9I, Cfr13I, Sau96I, Bsp1720I, BlpI, Bpu1102I, CelII, Bst4CI, BstDEI, DdeI, CpoI, CspI, RsrII, DsaI, BstDSI, Eco24I, BanII, EcoT38I, FriOI, HgiJII, Eco130I, StyI, BssT1I, EcoT14I, ErhI, EspI, BlpI, Bpu1102I, Bsp1720I, CelII, HgiAI, BsiHKAI, Alw21I, AspHI, Bbv12I, HinfI, PspPPI, PpuMI, Psp51I, SanDI, SduI, Bsp1286I, BmyI, SecI, BsaJI, BseDI, SfcI, BfmI, BstSFI, SmlI, or a restriction enzyme which has the same recognition site as AvaI, Ama87I, BcoI, BsoBI, Eco88I, AvaII, Eco47I, Bme18I, HgiEI, SinI, BanI, AccB1I, BshNI, Eco64I, BfmI, BstSFI, SfcI, Bpu10I, BsaMI, BscCI, BsmI, Mva1269I, Bsh12851, BsaOI, BsiEI, BstMCI, Bse1I, BseNI, BsrI, Cfr10I, BsiI, BssSI, Bst2BI, BsiZI, AspS9I, Cfr13I, Sau96I, Bsp1720I, BlpI, Bpu1102I, CelII, Bst4CI, BstDEI, DdeI, CpoI, CspI, RsrII, DsaI, BstDSI, Eco24I, BanII, EcoT38I, FriOI, HgiJII, Eco130I, StyI, BssT1I, EcoT14I, ErhI, EspI, BlpI, Bpu1102I, Bsp1720I, CelII, HgiAI, BsiHKAI, Alw21I, AspHI, Bbv12I, HinfI, PspPPI, PpuMI, Psp5II, SanDI, SduI, Bsp1286I, BmyI, SecI, BsaJI, BseDI, SfcI, BfmI, BstSFI, SmlI.  
     
     
         36 . The method of  claim 1  or  28  wherein one of the restriction enzymes is AarI, AscI, BbrCI, CspI, Dral, FseI, NotI, NruI, PacI, PmeI, PvuI, SapI, SdaI, SfiI, SgfI, SplI, SrfI, SwaI, or a restriction enzyme that has the same recognition site as AarI, AscI, BbrCI, CspI, DraI, FseI, NotI, NruI, PacI, PmeI, PvuI, SapI, SdaI, SfiI, SgfI, SplI, SrfI, SwaI.  
     
     
         37 . A method of inducing expression of a DNA sequence of interest in a host cell, comprising contacting a recombinant host cell which is deficient in rhamnose catabolism, and has a recombinant DNA molecule comprising a rhamnose-inducible promoter operably linked to an open reading frame for a heterologous RNA polymerase, with rhamnose and an expression vector comprising a promoter for the heterologous RNA polymerase operably linked to a DNA sequence of interest.  
     
     
         38 . The method of  claim 37  wherein the DNA sequence of interest is flanked by two restriction enzyme sites, wherein one of the flanking restriction enzyme sites is for a first restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates single-strand DNA overhangs, and wherein another flanking restriction enzyme site is for a second restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates ends that are not complementary to the overhangs generated by the first restriction enzyme.  
     
     
         39 . A method comprising introducing a vector comprising a nucleic acid fragment encoding a barnase which lacks a secretory domain into a recombinant host cell which expresses barstar from a promoter which is consitutively expressed in prokaryotic cells.  
     
     
         40 . A method comprising introducing the vector system of  claim 14  into a host cell, wherein the second vector comprises a counterselectable gene comprising a nucleic acid fragment encoding a barnase which lacks a secretory domain.  
     
     
         41 . A vector comprising an open reading frame 3′ to a DNA fragment of no more than 30 base pairs, which DNA fragment comprises a ribosome binding site, a SgfI recognition site, and a sequence which, when present in mRNA, enhances the binding of the mRNA to the small subunit of a eukaryotic ribosome.  
     
     
         42 . The vector of  claim 41  wherein the DNA fragment includes AAGGAGCGATCGCX 1 ATGX 2  (SEQ ID NO:1), and wherein X 1  and X 2  are individually an A, T, G or C.  
     
     
         43 . A vector comprising a SgfI recognition site, a sequence which comprises ATG and which sequence, when present in mRNA, enhances the binding of the mRNA to the small subunit of a eukaryotic ribosome, and an open reading frame which begins at the ATG in the sequence.  
     
     
         44 . A vector comprising a SgfI recognition site 5′ to a recognition site for a first restriction enzyme which generates blunt ends, which vector, once digested with SgfI and the first restriction enzyme and ligated to a DNA fragment comprising an open reading frame flanked by an end generated by a second restriction enzyme that generates a 3′ TA overhang and an end generated by a third restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates blunt ends, yields a recombinant vector comprising the open reading frame.  
     
     
         45 . The vector of  claim 44  wherein the first and third restriction enzymes are the same.  
     
     
         46 . The vector of  claim 44  wherein the first and third restriction enzymes are different.  
     
     
         47 . The vector of  claim 44  wherein the first restriction enzyme is PmeI, EcoRV or BalI.  
     
     
         48 . The vector of  claim 44  wherein the first restriction enzyme is PmeI, DraI, EsaBC3I, HindIII, HpaI, SciI or SwaI.  
     
     
         49 . The vector of  claim 44  wherein the first restriction enzyme is AluI, BalI, BfrBI, BsaAI, BsaBI, BsrBI, BtrI, Cac8I, CdiI, CviJI, CviRI, Eco47III, Eco78I, EcoICRI, EcoRV, FnuDII, FspAI, HaeI, HaeIII, Hpy8I, LpnI, MlyI, MslI, MstI, NaeI, NalIV, NruI, NspBII, OliI, PmaCI, PmeI, PshAI, PsiI, PvuII, RsaI, ScaI, SmaI, SnaBI, SrfI, SspI, SspD5I, StuI, XcaI, XmnI, or ZraI.  
     
     
         50 . The vector of  claim 44  wherein the restriction enzyme that generates a 3′ TA overhang is SgfI.  
     
     
         51 . The vector of  claim 44  which further comprises an open reading frame which includes the SgfI site.  
     
     
         52 . The vector of  claim 44  which comprises a ribosome binding site 5′ to the nucleotide cleaved by SgfI.  
     
     
         53 . The vector of  claim 44  wherein ligation generates the following sequence in the recombinant vector AAGGAGCGATCGCYATG or X 1 X 2 X 3 GCGATCGCCATG, wherein X 1 -X 3 , X 2 X 3 G or X 3 GC is a codon which is not a stop codon, and wherein Y is A, T, G or C.  
     
     
         54 . The vector of  claim 44  wherein ligation generates the following sequence in the recombinant vector X 1 X 2 X 3 GTTTY 1 Y 2 , wherein X 1 X 2 X 3  is a codon in an open reading frame which is not a stop codon and Y 1  and Y 2  each=A, Y 1 =A and Y 2 =G or Y 1 =G and Y 2 =A.  
     
     
         55 . The vector of  claim 44  wherein ligation generates the following sequence in the recombinant vector X 1 X 2 X 3 GTTTY 1 Y 2 , wherein X 1 X 2 X 3 , X 2 X 3 G or X 3 GT is a codon in an open reading frame which is not a stop codon and Y 1  is not A when Y 2  is A or G, or Y 1  is not G when Y 2  is A.  
     
     
         56 . A vector comprising a first open reading frame which includes a SgfI recognition site and a recognition site which is not in the open reading frame for a restriction enzyme that has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates blunt ends, which vector, once digested with SgfI and the restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates blunt ends, and ligated to a DNA fragment comprising a second open reading flanked by a single-strand 3′ TA DNA overhang and a blunt end, yields a recombinant vector comprising a third open reading frame comprising the first and second open reading frames, which third open reading frame encodes a fusion peptide or protein.  
     
     
         57 . A vector comprising a ribosome binding site which optionally overlaps by one nucleotide with a SgfI recognition site and a recognition site which is not in the open reading frame for a restriction enzyme that has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates blunt ends, which vector, once digested with SgfI and the restriction enzyme that has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates blunt ends, and ligated to a DNA fragment comprising an open reading frame encoding a peptide or polypeptide flanked by  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′ CGCCATGX 1 Y 1   
                     
                 
                     
                     
                 
                     
                   3′ TAGCGGTACX 2 Y 2   
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
       and a blunt end, yields a recombinant vector which encodes the peptide or polypeptide, wherein X 1  is the first codon which is 3′ to the start codon for the open reading frame, wherein X 2  is the complement of X 1 , wherein Y 1  is the remainder of the open reading frame, and wherein Y 2  is the complement of Y 1 .  
     
     
         58 . The vector of  claim 57  wherein X 1 =GR 1 R 2 , wherein R 1  or R 2 =A, T, C or G.  
     
     
         59 . A vector comprising a first open reading frame which includes a PmeI recognition site and is flanked at the 5′ end by a recognition site for a first restriction enzyme that generates complementary single-strand DNA overhangs, which vector, once digested with PmeI and the first restriction enzyme, and ligated to a DNA fragment comprising a blunt end at the 5′ end of a second open reading frame and an end generated by a second restriction enzyme which generates single-strand DNA overhangs which are complementary to the single-strand DNA overhangs generated by the first restriction enzyme, yields a recombinant vector comprising a third open reading frame comprising the first and second open reading frames.  
     
     
         60 . The vector of  claim 59  wherein the third open reading frame includes N 1 N 2 N 3 GTTTN 4 N 5 R, wherein N 1 N 2 N 3  and TN 4 N 5  are codons that do not code for a stop codon, and wherein R is one or more codons.  
     
     
         61 . The vector of  claim 59  wherein the blunt end of the DNA fragment is generated by a restriction enzyme other than PmeI.  
     
     
         62 . The vector of  claim 59  wherein the blunt end of the DNA fragment is generated by PmeI digestion.  
     
     
         63 . A vector comprising a first open reading frame which includes a PmeI recognition site and is flanked at the 5′ end by site for a first restriction enzyme that generates complementary single-strand DNA overhangs, which vector, once digested with PmeI and the first restriction enzyme, and ligated to a DNA fragment comprising a blunt end and an end generated by a second restriction enzyme which generates single-strand DNA overhangs which are complementary to the single-strand DNA overhangs generated by the first restriction enzyme, yields a recombinant vector which includes N 1 N 2 N 3 GTTTN 4 N 5 , wherein N 1 N 2 N 3 GTTT is a sequence from the 3′ end of the digested expression vector, wherein N 1 N 2 N 3  do not code for a stop codon, and wherein N 4  and N 5 =A, or N 4 =A and N 5 =G or N 4 =G and N 5 =A.  
     
     
         64 . The vector of  claim 63  wherein the blunt end of the DNA fragment is generated by PmeI digestion.  
     
     
         65 . The vector of  claim 63  wherein the blunt end of the DNA fragment is generated by a restriction enzyme other than PmeI.  
     
     
         66 . A method for performing genetic analysis, comprising: 
 a) populating a database of genetic data with a plurality of genetic records;    b) querying the database of genetic data to identify a first subset of genetic records, wherein each record has at least one recognition site for one predetermined restriction enzyme or for restriction enzymes included in a set of predetermined restriction enzymes; and    c) determining a set of statistics associated with the restriction enzyme recognition sites for at least a second subset of genetic records in the first subset.    
     
     
         67 . The method of  claim 66  wherein determining the set of statistics includes determining a number of genetic records including recognition sites for one predetermined restriction enzyme or for each of the predetermined restriction enzymes in the set.  
     
     
         68 . The method of  claim 66  wherein determining the set of statistics includes determining a number of occurrences of at least one site for the one predetermined restriction enzyme or for the predetermined restriction enzymes in a genetic record in the second subset.  
     
     
         69 . The method of  claim 66  wherein the genetic records comprise nucleic acid sequences.  
     
     
         70 . The method of  claim 66  further comprising filtering the subset of genetic records to include or exclude genetic records having one or more selected characteristics.  
     
     
         71 . The method of  claim 66  further comprising filtering the subset of genetic records to exclude genetic records having a size greater than a predetermined value.  
     
     
         72 . The method of  claim 71  wherein the predetermined value is 21000 characters.  
     
     
         73 . The method of  claim 66  further comprising determining the sequence of specific bases which are present as ambiguous bases within a recognition site or which are present between a recognition site for a restriction enzyme and the position at which the restriction enzyme cleaves DNA containing the recognition site.  
     
     
         74 . The method of  claim 66  wherein at least one of the restriction enzymes has a 6 bp, 7 bp or 8 bp recognition site.  
     
     
         75 . The method of  claim 66  wherein at least one of the restriction enzymes is a hapaxoterministic restriction enzyme.  
     
     
         76 . A computerized system for genetic analysis, comprising: 
 a database of genetic data;    a processor;    a set of one or more programs executed by the processor causing the processor to; 
 query the database of genetic data to identify a first subset of genetic records, wherein each record has at least one recognition site for one predetermined restriction enzyme or for restriction enzymes included in a set of predetermined restriction enzymes, and;  
 determine a set of statistics associated with the restriction enzyme recognition sites for at least a second subset of genetic records in the first subset.  
   
     
     
         77 . A recombinant vector prepared by digesting a vector comprising a SgfI recognition site 5′ to a recognition site for a first restriction enzyme which generates blunt ends, with SgfI and the first restriction enzyme and ligating the digested vector to a DNA fragment comprising an open reading frame flanked by an end generated by a second restriction enzyme that generates a 3′ TA overhang and an end generated by a third restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates blunt ends.  
     
     
         78 . A support comprising a plurality of recombinant vectors, one or more of which comprise a different open reading frame, wherein each recombinant vector is prepared by digesting a vector comprising a SgfI recognition site 5′ to a recognition site for a first restriction enzyme which generates blunt ends, with SgfI and the first restriction enzyme and ligating the digested vector to a DNA fragment comprising an open reading frame flanked by an end generated by a second restriction enzyme that generates a 3′ TA overhang and an end generated by a third restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates blunt ends.  
     
     
         79 . The support of  claim 78  which a multi-well plate, the wells of which optionally each comprise a different recombinant vector.  
     
     
         80 . The support of  claim 78  wherein the different open reading frames include open reading frames having nucleotide substitutions of a selected open reading frame which different open reading frames are prepared by mutatgenesis of the selected open reading frame.  
     
     
         81 . A vector comprising a SgfI recognition site 5′ to an opening reading frame which begins with an ATG and ends with an in-frame stop codon provided by nucleotides TAA in a PmeI recognition site.  
     
     
         82 . A vector prepared by ligating 
 a DNA fragment comprising an opening reading frame which begins with an ATG but has no in-frame stop codon, a 5′ end generated after cleavage of a first recognition site with a first restriction enzyme which generates an end compatible with an end generated after cleavage of a SgfI recognition site by SgfI, and a 3′ end generated after cleavage of a second recognition site with a second restriction enzyme which generates a blunt end, and    a DNA segment comprising a 5′ end generated after cleavage of PmeI recognition site by PmeI and a 3′ end generated after cleavage of a third recognition site with a third enzyme which generates an end compatible with an end generated after cleavage of a SgfI recognition site by SgfI, wherein the first recognition site is cleaved by SgfI, the third recognition site is cleaved by SgfI, or both the first and third recognition sites are cleaved by SgfI, and wherein the TAA in the PmeI recognition site provides an in-frame stop codon for the open reading frame.    
     
     
         83 . The vector of  claim 81  wherein the SgfI recognition site and the ATG for the open reading frame comprise GCGATCGCNATGG, wherein n is C, A, T or G.  
     
     
         84 . The vector of  claim 82  wherein the first and third recognition sites are cleaved by SgfI and yield an exchange site comprising GCGATCGCnATGG, wherein n is C, A, T or G.  
     
     
         85 . The vector of  claim 83  or  84  wherein n is C.  
     
     
         86 . A vector comprising a SgfI recognition site 5′ to an opening reading frame which begins with an ATG and ends with a stop codon that is 5′ to a PmeI recognition site.  
     
     
         87 . A vector prepared by ligating 
 a DNA fragment comprising an opening reading frame which begins with an ATG and ends with a stop codon, a 5′ end generated after cleavage of a first recognition site with a first restriction enzyme which generates an end compatible with an end generated after cleavage of a SgfI recognition site by SgfI, and a 3′ end generated after cleavage of a second recognition site for a second restriction enzyme which generates a blunt end, and    a DNA segment comprising a 5′ end generated after cleavage of a third recognition site for a third restriction enzyme which generates a blunt end and a 3′ end generated after cleavage of a fourth recognition site with a fourth restriction enzyme which generates an end compatible with an end generated after cleavage of a SgfI recognition site by SgfI, wherein the first recognition site is cleaved by SgfI, the fourth recognition site is cleaved by SgfI, or both the first and fourth recognition sites are cleaved by SgfI, and wherein the second recognition site is cleaved by PmeI, the third recognition site is cleaved by PmeI or both the second and third recognition sites are cleaved by PmeI.    
     
     
         88 . The vector of  claim 81  or  86  which further comprises a promoter 5′ to the SgfI site.  
     
     
         89 . The vector of  claim 82  or  87  which further comprises a promoter 5′ to the exchange site formed by ligation of the SgfI compatible ends.  
     
     
         90 . The vector of  claim 86  wherein the SgfI recognition site and the ATG for the open reading frame comprise GCGATCGCNATG, wherein n is C, A, T or G.  
     
     
         91 . The vector of  claim 87  wherein the first and fourth recognition sites are cleaved by SgfI and yield an exchange site comprising GCGATCGCnATG, wherein n is C, A, T or G.  
     
     
         92 . The vector of  claim 90  or  91  wherein n is C.  
     
     
         93 . A vector comprising a SgfI recognition site 5′ to an opening reading frame which does not begin with an ATG but ends with an in-frame stop codon provided by nucleotides TAA in a PmeI recognition site.  
     
     
         94 . A vector encoding a fusion polypeptide prepared by ligating 
 a DNA fragment comprising a first opening reading frame which does not begin with an ATG, a 5′ end generated after cleavage of a first recognition site with a first restriction enzyme which generates an end compatible with an end generated after cleavage of a SgfI recognition site by SgfI, and a 3′ end generated after cleavage of a second recognition site for a second restriction enzyme which generates a blunt end, and    a DNA segment comprising a second open reading frame that begins with an ATG but does not end with an in-frame stop codon, a 3′ end generated after cleavage of a third recognition site with a third restriction enzyme which generates an end compatible with an end generated after cleavage of a SgfI recognition site by SgfI, and a 5′ end generated after cleavage of a PmeI site with PmeI, wherein the first recognition site is cleaved by SgfI, the third recognition site is cleaved by SgfI, or both the first and third recognition sites are cleaved by SgfI, wherein the TAA in the PmeI site provides an in-frame stop codon for the first open reading frame, and wherein ligation of the SgfI compatible ends yields a third open reading frame comprising the first and second open reading frames which encodes the fusion polypeptide.    
     
     
         95 . The vector of  claim 94  wherein the DNA segment further comprises a promoter 5′ to the ATG of the second open reading frame.  
     
     
         96 . A vector comprising a SgfI recognition site 5′ to an opening reading frame which includes sequences for a domain, which open reading frame does not begin with an ATG and ends with a stop codon that is 5′ to a PmeI recognition site.  
     
     
         97 . A vector encoding a fusion polypeptide prepared by ligating 
 a DNA fragment comprising a first opening reading frame which includes sequences for a domain which first open reading frame does not begin with an ATG and ends with a stop codon, a 5′ end generated after cleavage of a first recognition site with a first restriction enzyme which generates an end compatible with an end generated after cleavage of a SgfI recognition site by SgfI, and a 3′ end generated after cleavage of a second recognition site for a second restriction enzyme which generates a blunt end, and    a DNA segment comprising a second open reading frame that begins with an ATG but does not end with an in-frame stop codon, a 3′ end generated after cleavage of a third recognition site with a third restriction enzyme which generates an end compatible with an end generated after cleavage of a SgfI recognition site by SgfI, and a 5′ end generated after cleavage of a fourth recognition site with a fourth restriction enzyme that generates a blunt end, wherein the first recognition site is cleaved by SgfI, the third recognition site is cleaved by SgfI or both the first and third recognition sites are cleaved by SgfI, and wherein the second recognition site is cleaved by PmeI, the fourth recognition site is cleaved by PmeI or both the second and fourth recognition sites are cleaved by PmeI, and wherein ligation of the SgfI compatible ends yields a third open reading frame comprising the first and second open reading frames which encodes the fusion polypeptide.    
     
     
         98 . The vector of  claim 97  wherein the DNA segment further comprising a promoter 5′ to the ATG of the second open reading frame.