COMPOSITIONS AND METHODS FOR USE IN RECOMBINATIONAL CLONING OF NUCLEIC ACIDS

Abstract: The present invention relates generally to compositions and methods for use in recombinational cloning of nucleic acid molecules. In particular, the invention relates to nucleic acid molecules encoding one or more recombination sites or portions thereof, to nucleic acid molecules comprising one or more of these recombination site nucleotide sequences and optionally comprising one or more additional physical or functional nucleotide sequences. The invention also relates to vectors comprising the nucleic acid molecules of the invention, to host cells comprising the vectors or nucleic acid molecules of the invention, to methods of producing polypeptides using the nucleic acid molecules of the invention, and to polypeptides encoded by these nucleic acid molecules or produced by the methods of the invention. The invention also relates to antibodies that bind to one or more polypeptides of the invention or epitopes thereof. The invention also relates to the use of these compositions in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and/or DNA segments.

Description: Compositions and Methods for Use in Recombinational Cloning of Nucleic Acids BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to recombinant DNA technology More particularly, the present invention relates to compositions and methods for use in recombinational cloning of nucleic acid molecules The invention relates specifically to nucleic acid molecules encoding one or more recombination sites or one or more partial recombination sites, particularly αttB, αttP, αttL, and αttR, and fragments, mutants, variants and derivatives thereof The invention also relates to such nucleic acid molecules wherein the one or more recombination site nucleotide sequences is operably linked to the one or more additional physical or functional nucleotide sequences The invention also relates to vectors comprising the nucleic acid molecules of the invention, to host cells comprising the vectors or nucleic acid molecules of the invention, to methods of producing polypeptides and RNAs encoded by the nucleic acid molecules of the invention, and to polypeptides encoded by these nucleic acid molecules or produced by the methods of the invention, which may be fusion proteins The invention also relates to antibodies that bind to one or more polypeptides of the invention or epitopes thereof, which may be monoclonal or polyclonal antibodies The invention also relates to the use of these nucleic acid molecules, vectors, polypeptides and antibodies in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and/or DNA segments More particularly, the antibodies of the invention may be used to identify and/or purify proteins or fusion proteins encoded by the nucleic acid molecules or vectors of the invention, or to identify and/or purify the nucleic acid molecules of the invention RelatedArt Site-specific recombinases Site-specific recombinases are proteins that are present in many organisms (e g viruses and bacteria) and have been characterized to have both endonuclease and ligase properties These recombinases (along with associated proteins in some cases) recognize specific sequences of bases in DNA and exchange the DNA segments flanking those segments The recombinases and associated proteins are collectively referred to as "recombination proteins" (see, e g , Landy, A , Current Opinion in Biotechnology 3 699-707 (1993)) Numerous recombination systems from various organisms have been described See, e.g., Hoess et al., Nucleic Acids Research 14(6) 2287 (1986), Abremski et al, J Biol Chem.261(1) 391 (1986), Campbell, J. Bacterwl. 174(23) 7495 (1992), Qian etal.,J. Biol. Chem. 267(11) 7794 (1992), Araki et al, J. Mol. Biol 225(1) 25 (1992), Maeser and Kahnmann Mol. Gen. Genet. 230 170-176) (1991), Esposito et al , Nucl. Acids Res. 25(18) 3605 (1997) Many of these belong to the integrase family of recombinases (Argos et al. EMBOJ. 5 433-440 (1986), Voziyanov etal, Nucl. Acids Res. 27 930 (1999)) Perhaps the best studied of these are the Integrase/αtt system from bacteriophage λ (Landy, A Current Opinions in Genetics andDevel. 3 699-707 ( 1993)), the CvdloxP system from bacteriophage P 1 (Hoess and Abremski ( 1990) InNucleic Acids and Molecular Biology, vol 4 Eds Eckstein and Lilley, Berlin- Heidelberg Springer- Verlag, pp 90-109) , and the FLP/FRT system from the Saccharomyces cerevisiae 2 μ circle plasmid (Broach et al. Cell 29 227-234 (1982)) Backman (U S Patent No 4,673,640) discloses the in vivo use of λ recombinase to recombine a protein producing DNA segment by enzymatic site- specific recombination using wild-type recombination sites attB and attP Hasan and Szybalski (Gene 56 145-151 (1987)) discloses the use of λ Int recombinase in vivo for intramolecular recombination between wild type attP and attB sites which flank a promoter Because the orientations of these sites are inverted relative to each other, this causes an irreversible flipping of the promoter region relative to the gene of interest Palazzolo et al. Gene 88 25-36 (1990), discloses phage lambda vectors having bacteriophage λ arms that contain restriction sites positioned outside a cloned DNN sequence and between wild-type loxP sites Infection of E. coli cells that express the Cre recombinase with these phage vectors results in recombination between the loxP sites and the in vivo excision of the plasmid replicon, including the cloned cDΝA Pόsfai et al. (Nucl. Acids Res. 22 2392-2398 (1994)) discloses a method for inserting into genomic DΝA partial expression vectors having a selectable marker, flanked by two wild-type FRT recognition sequences FLP site-specific recombinase as present in the cells is used to integrate the vectors into the genome at predetermined sites Under conditions where the replicon is functional, this cloned genomic DΝA can be amplified Bebee etal. (U S Patent No 5,434,066) discloses the use of site-specific recombinases such as Cre for DNA containing two loxP sites for in vivo recombination between the sites Boyd (Nucl. Acids Res. 21 817-821 (1993)) discloses a method to facilitate the cloning of blunt-ended DNA using conditions that encourage intermolecular ligation to a dephosphorylated vector that contains a wild-type loxP site acted upon by a Cre site-specific recombinase present in E. coli host cells Waterhouse et al. (WO 93/19172 and Nucleic Acids Res. 21 (9)2265 (1993)) disclose an in vivo method where light and heavy chains of a particular antibody were cloned in different phage vectors between loxP and loxP 511 sites and used to transfect new E. coli cells Cre, acting in the host cells on the two parental molecules (one plasmid, one phage), produced four products in equilibrium two different cointegrates (produced by recombination at either loxP or loxP 511 sites), and two daughter molecules, one of which was the desired product Schlake & Bode (Biochemistry 33 12746-12751 (1994)) discloses an in vivo method to exchange expression cassettes at defined chromosomal locations, each flanked by a wild type and a spacer-mutated FRT recombination site A double-reciprocal crossover was mediated in cultured mammalian cells by using this FLP/FRT system for site-specific recombination Hartley et al. (U S Patent No 5,888,732) disclose compositions and methods for recombinational exchange of nucleic acid segments and molecules, including for use in recombinational cloning of a variety of nucleic acid molecules in vitro and in vivo, using a combination of wildtype and mutated recombination sites and recombination proteins Transposases. The family of enzymes, the transposases, has also been used to transfer genetic information between replicons Transposons are structurally variable, being described as simple or compound, but typically encode the recombinase gene flanked by DNA sequences organized in inverted orientations Integration of transposons can be random or highly specific Representatives such as Tn7, which are highly site-specific, have been applied to the in vivo movement of DNA segments between replicons (Lucklow et al. , J. Virol. 67 4566-4579 (1993)). Devine and Boeke Nucl. Acids Res. 22 3765-3772 (1994), discloses the construction of artificial transposons for the insertion of DNA segments, in vitro, into recipient DNA molecules The system makes use of the integrase of yeast TYl virus-like particles The DNA segment of interest is cloned, using standard methods, between the ends of the transposon-like element TYl In the presence of the TYl integrase, the resulting element integrates randomly into a second target DNA molecule Recombination Sites. Also key to the integration/recombination reactions mediated by the above-noted recombination proteins and/or transposases are recognition sequences, often termed "recombination sites, " on the DNA molecules participating in the integration/recombination reactions These recombination sites are discrete sections or segments of DNA on the participating nucleic acid molecules that are recognized and bound by the recombination proteins during the initial stages of integration or recombination For example, the recombination site for Cre recombinase is loxP which is a 34 base pair sequence comprised of two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence See Figure 1 of Sauer, B , Curr. Opin. Biotech. 5 521-527 (1994) Other examples of recognition sequences include the attB, attP, attL, and attR sequences which are recognized by the recombination protein A Int attB is an approximately 25 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region, while attP is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis) See Landy, Curr. Opin. Biotech. 3 699-707 (1993), see also U S Patent No 5,888,732, which is incorporated by reference herein DNA cloning. The cloning of DNA segments currently occurs as a daily routine in many research labs and as a prerequisite step in many genetic analyses The purpose of these clonings is various, however, two general purposes can be considered (1) the initial cloning of DNA from large DNA or RNA segments (chromosomes, YACs, PCR fragments, mRNA, etc ), done in a relative handful of known vectors such as pUC, pGem, pBlueScript, and (2) the subcloning of these DNA segments into specialized vectors for functional analysis A great deal of time and effort is expended both in the transfer of DNA segments from the initial cloning vectors to the more specialized vectors This transfer is called subcloning The basic methods for cloning have been known for many years and have changed little during that time A typical cloning protocol is as follows ( 1 ) digest the DNA of interest with one or two restriction enzymes, (2) gel purify the DNA segment of interest when known, (3) prepare the vector by cutting with appropriate restriction enzymes, treating with alkaline phosphatase, gel purify etc , as appropriate, (4) ligate the DNA segment to the vector, with appropriate controls to eliminate background of uncut and self-ligated vector, (5) introduce the resulting vector into an E. coli host cell, (6) pick selected colonies and grow small cultures overnight, (7) make DNA minipreps, and (8) analyze the isolated plasmid on agarose gels (often after diagnostic restriction enzyme digestions) or by PCR The specialized vectors used for subcloning DNA segments are functionally diverse These include but are not limited to vectors for expressing nucleic acid molecules in various organisms, for regulating nucleic acid molecule expression, for providing tags to aid in protein purification or to allow tracking of proteins in cells, for modifying the cloned DNA segment (e.g., generating deletions), for the synthesis of probes (e.g., riboprobes), for the preparation of templates for DNA sequencing, for the identification of protein coding regions, for the fusion of various protein-coding regions, to provide large amounts of the DNA of interest, etc It is common that a particular investigation will involve subcloning the DNA segment of interest into several different specialized vectors As known in the art, simple subclonings can be done in one day (e.g., the DNA segment is not large and the restriction sites are compatible with those of the subcloning vector) However, many other subclonings can take several weeks, especially those involving unknown sequences, long fragments, toxic genes, unsuitable placement of restriction sites, high backgrounds, impure enzymes, etc Subcloning DNA fragments is thus often viewed as a chore to be done as few times as possible Several methods for facilitating the cloning of DNA segments have been described, e.g., as in the following references Ferguson, J , et al. Gene 16 191 (1981), discloses a family of vectors for subcloning fragments of yeast DNA The vectors encode kanamycin resistance Clones of longer yeast DNA segments can be partially digested and ligated into the subcloning vectors If the original cloning vector conveys resistance to ampicillin, no purification is necessary prior to transformation, since the selection will be for kanamycin Hashimoto-Gotoh, T , et al. Gene 41 125 (1986), discloses a subcloning vector with unique cloning sites within a streptomycin sensitivity gene, in a streptomycin-resistant host, only plasmids with inserts or deletions in the dominant sensitivity gene will survive streptomycin selection Accordingly, traditional subcloning methods, using restriction enzymes and ligase, are time consuming and relatively unreliable Considerable labor is expended, and if two or more days later the desired subclone can not be found among the candidate plasmids, the entire process must then be repeated with alternative conditions attempted Although site specific recombinases have been used to recombine DNA in vivo, the successful use of such enzymes in vitro was expected to suffer from several problems For example, the site specificities and efficiencies were expected to differ in vitro, topologically linked products were expected, and the topology of the DNA substrates and recombination proteins was expected to differ significantly in vitro (see, e.g., Adams et al, J. Mol. Biol 226 661-73 (1992)) Reactions that could go on for many hours in vivo were expected to occur in significantly less time in vitro before the enzymes became inactive In addition, the stabilities of the recombination enzymes after incubation for extended periods of time in in vitro reactions was unknown, as were the effects of the topologies (i.e., linear, coiled, supercoiled, etc ) of the nucleic acid molecules involved in the reaction Multiple DNA recombination products were expected in the biological host used, resulting in unsatisfactory reliability, specificity or efficiency of subcloning Thus, in vitro recombination reactions were not expected to be sufficiently efficient to yield the desired levels of product Accordingly, there is a long felt need to provide an alternative subcloning system that provides advantages over the known use of restriction enzymes and ligases SUMMARY OF THE INVENTION The present invention relates to nucleic acid molecules encoding one or more recombination sites or one or more partial recombination sites, particularly αttB, αttP, ttL, and attR, and fragments, mutants, variants and derivatives thereof The invention also relates to such nucleic acid molecules comprising one or more of the recombination site nucleotide sequences or portions thereof and one or more additional physical or functional nucleotide sequences, such as those encoding one or more multiple cloning sites, one or more transcription termination sites, one or more transcriptional regulatory sequences (e.g., one or more promoters, enhancers, or repressors), one or more translational signal sequences, one or more nucleotide sequences encoding a fusion partner protein or peptide (e-g-, GST, His 6 or thioredoxin), one or more selection markers or modules, one or more nucleotide sequences encoding localization signals such as nuclear localization signals or secretion signals, one or more origins of replication, one or more protease cleavage sites, one or more desired proteins or peptides encoded by a gene or a portion of a gene, and one or more 5' or 3' polynucleotide tails (particularly a poly-G tail). The invention also relates to such nucleic acid molecules wherein the one or more recombination site nucleotide sequences is operably linked to the one or more additional physical or functional nucleotide sequences. The invention also relates to primer nucleic acid molecules comprising the recombination site nucleotide sequences of the invention (or portions thereof), and to such primer nucleic acid molecules linked to one or more target-specific (e.g. , one or more gene-specific) primer nucleic acid sequences. Such primers may also comprise sequences complementary or homologous to DNA or RNA sequences to be amplified, e.g., by PCR, RT-PCR, etc. Such primers may also comprise sequences or portions of sequences useful in the expression of protein genes (ribosome binding sites, localization signals, protease cleavage sites, repressor binding sites, promoters, transcription stops, stop codons, etc.). Said primers may also comprise sequences or portions of sequences useful in the manipulation of DNA molecules (restriction sites, transposition sites, sequencing primers, etc.). The primers of the invention may be used in nucleic acid synthesis and preferably are used for amplification (e.g., PCR) of nucleic acid molecules. When the primers of the invention include target- or gene-specific sequences (any sequence contained within the target to be synthesized or amplified including translation signals, gene sequences, stop codons, transcriptional signals (e.g., promoters) and the like), amplification or synthesis of target sequences or genes may be accomplished. Thus, the invention relates to synthesis of a nucleic acid molecules comprising mixing one or more primers of the invention with a nucleic acid template, and incubating said mixture under conditions sufficient to make a first nucleic acid molecule complementary to all or a portion of said template Thus, the invention relates specifically to a method of synthesizing a nucleic acid molecule comprising (a) mixing a nucleic acid template with a polypeptide having polymerase activity and one or more primers comprising one or more recombination sites or portions thereof, and (b) incubating said mixture under conditions sufficient to synthesize a first nucleic acid molecule complementary to all or a portion of said template and which preferably comprises one or more recombination sites or portions thereof Such method of the invention may further comprise incubating said first synthesized nucleic acid molecule under conditions sufficient to synthesize a second nucleic acid molecule complementary to all or a portion of said first nucleic acid molecule Such synthesis may provide for a first nucleic acid molecule having a recombination site or portion thereof at one or both of its termini In a preferred aspect, for the synthesis of the nucleic acid molecules, at least two primers are used wherein each primer comprises a homologous sequence at its terminus and/or within internal sequences of each primer (which may have a homology length of about 2 to about 500 bases, preferably about 3 to about 100 bases, about 4 to about 50 bases, about 5 to about 25 bases and most preferably about 6 to about 18 base overlap) In a preferred aspect, the first such primer comprises at least one target-specific sequence and at least one recombination site or portion thereof while the second primer comprises at least one recombination site or portion thereof Preferably, the homologous regions between the first and second primers comprise at least a portion of the recombination site In another aspect, the homologous regions between the first and second primers may comprise one or more additional sequences, e.g., expression signals, translational start motifs, or other sequences adding functionality to the desired nucleic acid sequence upon amplification In practice, two pairs of primers prime synthesis or amplification of a nucleic acid molecule In a preferred aspect, all or at least a portion of the synthesized or amplified nucleic acid molecule will be homologous to all or a portion of the template and further comprises a recombination site or a portion thereof at at least one terminus and preferably both termini of the synthesized or amplified molecule. Such synthesized or amplified nucleic acid molecule may be double stranded or single stranded and may be used in the recombinational cloning methods of the invention The homologous primers of the invention provide a substantial advantage in that one set of the primers may be standardized for any synthesis or amplification reaction That is, the primers providing the recombination site sequences (without the target specific sequences) can be pre-made and readily available for use This in practice allows the use of shorter custom made primers that contain the target specific sequence needed to synthesize or amplify the desired nucleic acid molecule Thus, this provides reduced time and cost in preparing target specific primers (e.g , shorter primers containing the target specific sequences can be prepared and used in synthesis reactions). The standardized primers, on the other hand, may be produced in mass to reduce cost and can be readily provided (e.g , in kits or as a product) to facilitate synthesis of the desired nucleic acid molecules Thus, in one preferred aspect, the invention relates to a method of synthesizing or amplifying one or more nucleic acid molecules comprising (a) mixing one or more nucleic acid templates with at least one polypeptide having polymerase or reverse transcriptase activity and at least a first primer comprising a template specific sequence (complementary to or capable of hybridizing to said templates) and at least a second primer comprising all or a portion of a recombination site wherein said at least a portion of said second primer is homologous to or complementary to at least a portion of said first primer, and (b) incubating said mixture under conditions sufficient to synthesize or amplify one or more nucleic acid molecules complementary to all or a portion of said templates and comprising one or more • recombination sites or portions thereof at one and preferably both termini of said molecules More specifically, the invention relates to a method of synthesizing or amplifying one or more nucleic acid molecules comprising: (a) mixing one or more nucleic acid templates with at least one polypeptide having polymerase or reverse transcriptase activity and at least a first primer comprising a template specific sequence (complementary to or capable of hybridizing to said templates) and at least a portion of a recombination site, and at least a second primer comprising all or a portion of a recombination site wherein said at least a portion of said recombination site on said second primer is complementary to or homologous to at least a portion of said recombination site on said first primer; and (b) incubating said mixture under conditions sufficient to synthesize or amplify one or more nucleic acid molecules complementary to all or a portion of said templates and comprising one or more recombination sites or portions thereof at one and preferably both termini of said molecules. In a more preferred aspect, the invention relates to a method of amplifying or synthesizing one or more nucleic acid molecules comprising: (a) mixing one or more nucleic acid templates with at least one polypeptide having polymerase or reverse transcriptase activity and one or more first primers comprising at least a portion of a recombination site and a template specific sequence (complementary to or capable of hybridizing to said template); (b) incubating said mixture under conditions sufficient to synthesize or amplify one or more first nucleic acid molecules complementary to all or a portion of said templates wherein said molecules comprise at least a portion of a recombination site at one and preferably both termini of said molecules; (c) mixing said molecules with one or more second primers comprising one or more recombination sites, wherein said recombination sites of said second primers are homologous to or complementary to at least a portion of said recombination sites on said first nucleic acid molecules, and (d) incubating said mixture under conditions sufficient to synthesize or amplify one or more second nucleic acid molecules complementary to all or a portion of said first nucleic acid molecules and which comprise one or more recombination sites at one and preferably both termini of said molecules The invention also relates to vectors comprising the nucleic acid molecules of the invention, to host cells comprising the vectors or nucleic acid molecules of the invention, to methods of producing polypeptides encoded by the nucleic acid molecules of the invention, and to polypeptides encoded by these nucleic acid molecules or produced by the methods of the -invention, which may be fusion proteins The invention also relates to antibodies that bind to one or more polypeptides of the invention or epitopes thereof, which may be monoclonal or polyclonal antibodies The invention also relates to the use of these nucleic acid molecules, primers, vectors, polypeptides and antibodies in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and/or DNA segments The antibodies of the invention may have particular use to identify and/or purify peptides or proteins (including fusion proteins produced by the invention), and to identify and/or purify the nucleic acid molecules of the invention or portions thereof The methods for in vitro or in vivo recombinational cloning of nucleic acid molecule generally relate to recombination between at least a first nucleic acid molecule having at least one recombination site and a second nucleic acid molecule having at least one recombination site to provide a chimeric nucleic acid molecule In one aspect, the methods relate to recombination between and first vector having at least one recombination site and a second vector having at least one recombination site to provide a chimeric vector In another aspect, a nucleic acid molecule having at least one recombination site is combined with a vector having at least one recombination site to provide a chimeric vector In a most preferred aspect, the nucleic acid molecules or vectors used in recombination comprise two or more recombination sites In a more specific embodiment of the invention, the recombination methods relate to a Destination Reaction (also referred to herein as an "LR reaction") in which recombination occurs between an Entry clone and a Destination Vector Such a reaction transfers the nucleic acid molecule of interest from the Entry Clone into the Destination Vector to create an Expression Clone The methods of the invention also specifically relate to an Entry or Gateward reaction (also referred to herein as a "BP reaction") in which an Expression Clone is recombined with a Donor vector to produce an Entry clone In other aspects, the invention relates to methods to prepare Entry clones by combining an Entry vector with at least one nucleic acid molecule (e g , gene or portion of a gene) The invention also relates to conversion of a desired vector into a Destination Vector by including one or more (preferably at least two) recombination sites in the vector of interest In a more preferred aspect, a nucleic acid molecule (e.g., a cassette) having at least two recombination sites flanking a selectable marker (e.g. , a toxic gene or a genetic element preventing the survival of a host cell containing that gene or element, and/or preventing replication, partition or heritability of a nucleic acid molecule (e.g., a vector or plasmid) comprising that gene or element) is added to the vector to make a Destination Vector of the invention Preferred vectors for use in the invention include prokaryotic vectors, eukaryotic vectors, or vectors which may shuttle between various prokaryotic and/or eukaryotic systems (e g shuttle vectors) Preferred prokaryotic vectors for use in the invention include but are not limited to vectors which may propagate and/or replicate in gram negative and/or gram positive bacteria, including bacteria ofthe genera Escherichiα, Salmonella, Proteus, Clostridium, Klebsiella, Bacillus, Streptomyces, andPseudomonas and preferably in the species E. coli Eukaryotic vectors for use in the invention include vectors which propagate and/or replicate and yeast cells, plant cells, mammalian cells, (particularly human and mouse), fungal cells, insect cells, nematode cells, fish cells and the like Particular vectors of interest include but are not limited to cloning vectors, sequencing vectors, expression vectors, fusion vectors, two-hybrid vectors, gene therapy vectors, phage display vectors, gene-targeting vectors, PACs, BACs, YACs, MACs, and reverse two-hybrid vectors. Such vectors may be used in prokaryotic and/or eukaryotic systems depending on the particular vector. In another aspect, the invention relates to kits which may be used in carrying out the methods of the invention, and more specifically relates to cloning or subcloning kits and kits for carrying out the LR Reaction (e.g., making an Expression Clone), for carrying out the BP Reaction (e.g., making an Entry Clone), and for making Entry Clone and Destination Vector molecules of the invention. Such kits may comprise a carrier or receptacle being compartmentalized to receive and hold therein any number of containers. Such containers may contain any number of components for carrying out the methods of the invention or combinations of such components. In particular, a kit of the invention may comprise one or more components (or combinations thereof) selected from the group consisting of one or more recombination proteins or auxiliary factors or combinations thereof, one or more compositions comprising one or more recombination proteins or auxiliary factors or combinations thereof (for example, GATEWAY™ LR Clonase™ Enzyme Mix or GATEWAY™ BP Clonase™ Enzyme Mix), one or more reaction buffers, one or more nucleotides, one or more primers of the invention, one or more restriction enzymes, one or more ligases, one or more polypeptides having polymerase activity (e.g., one or more reverse transcriptases or DNA polymerases), one or more proteinases (e.g. , proteinase K or other proteinases), one or more Destination Vector molecules, one or more Entry Clone molecules, one or more host cells (e.g. competent cells, such as E. coli cells, yeast cells, animal cells (including mammalian cells, insect cells, nematode cells, avian cells, fish cells, etc.), plant cells, and most particularly E. coli DB3.1 host cells, such as E. coli LIBRARY EFFICIENCY® DB3.1™ Competent Cells), instructions for using the kits of the invention (e.g. , to carry out the methods of the invention), and the like. In related aspects, the kits of the invention may comprise one or more nucleic acid molecules encoding one or more recombination sites or portions thereof, particularly one or more nucleic acid molecules comprising a nucleotide sequence encoding the one or more recombination sites or portions thereof of the invention. Preferably, such nucleic acid molecules comprise at least two recombination sites which flank a selectable marker (e.g. , a toxic gene and/or antibiotic resistance gene). In a preferred aspect, such nucleic acid molecules are in the form of a cassette (e.g. , a linear nucleic acid molecule comprising one or more and preferably two or more recombination sites or portions thereof). Kits for inserting or adding recombination sites to nucleic acid molecules of interest may comprise one or more nucleases (preferably restriction endonucleases), one or more ligases, one or more topoisomerases, one or more polymerases, and one or more nucleic acid molecules or adapters comprising one or more recombination sites. Kits for integrating recombination sites into one or more nucleic acid molecules of interest may comprise one or more components (or combinations thereof) selected from the group consisting of one or more integration sequences comprising one or more recombination sites. Such integration sequences may comprise one or more transposons, integrating viruses, homologous recombination sequences, RNA molecules, one or more host cells and the like. Kits for making the Entry Clone molecules of the invention may comprise any or a* number of components and the composition of such kits may vary depending on the specific method involved. Such methods may involve inserting the nucleic acid molecules of interest into an Entry or Donor Vector by the recombinational cloning methods of the invention, or using conventional molecular biology techniques (e.g. , restriction enzyme digestion and ligation). In a preferred aspect, the Entry Clone is made using nucleic acid amplification or synthesis products. Kits for synthesizing Entry Clone molecules from amplification or synthesis products may comprise one or more components (or combinations thereof) selected from the group consisting of one or more Donor Vectors (e.g., one or more attP vectors including, but not limited to, pDONR201 (Figure 49), pDONR202 (Figure 50), pDONR203 (Figure 51), pDONR204 (Figure 52), pDONR205 (Figure 53), pDONR206 (Figure 53), and the like), one or more polypeptides having polymerase activity (preferably DNA polymerases and most preferably thermostable DNA polymerases), one or more proteinases, one or more reaction buffers, one or more nucleotides, one or more primers comprising one or more recombination sites or portions thereof, and instructions for making one or more Entry Clones. Kits for making the Destination vectors of the invention may comprise any number of components and the compositions of such kits may vary depending on the specific method involved. Such methods may include the recombination methods of the invention or conventional molecular biology techniques (e.g., restriction endonuclease digestion and ligation). In a preferred aspect, the Destination vector is made by inserting a nucleic acid molecule comprising at least one recombination site (or portion thereof) of the invention (preferably a nucleic acid molecule comprising at least two recombination sites or portions thereof flanking a selectable marker) into a desired vector to convert the desired vector into a Destination vector of the invention. Such kits may comprise at least one component (or combinations thereof) selected from the group consisting of one or more restriction endonucleases, one or more ligases, one or more polymerases, one or more nucleotides, reaction buffers, one or more nucleic acid molecules comprising at least one recombination site or portion thereof (preferably at least one nucleic acid molecule comprising at least two recombination sites flanking at least one selectable marker, such as a cassette comprising at least one selectable marker such as antibiotic resistance genes and/or toxic genes), and instructions for making such Destination vectors. The invention also relates to kits for using the antibodies of the invention in identification and/or isolation of peptides and proteins (which may be fusion proteins) produced by the nucleic acid molecules of the invention, and for identification and/or isolation of the nucleic acid molecules of the invention or portions thereof. Such kits may comprise one or more components (or combination thereof) selected from the group consisting of one or more antibodies of the invention, one or more detectable labels, one or more solid supports and the like. Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of what is known in the art, in light of the following drawings and description of the invention, and in light of the claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts one general method of the present invention, wherein the starting (parent) DNA molecules can be circular or linear The goal is to exchange the new subcloning vector D for the original cloning vector B It is desirable in one embodiment to select for AD and against all the other molecules, including the Cointegrate The square and circle are sites of recombination e.g., lox (such as loxP) sites, att sites, etc. For example, segment D can contain expression signals, protein fusion domains, new drug markers, new origins of replication, or specialized functions for mapping or sequencing DNA It should be noted that the cointegrate molecule contains Segment D (Destination vector) adjacent to segment A (Insert), thereby juxtaposing functional elements in D with the insert in A Such molecules can be used directly in vitro (e.g. , if a promoter is positioned adjacent to a gene-for in vitro transcription/translation) or in vivo (following isolation in a cell capable of propagating ccdB-containing vectors) by selecting for the selection markers in Segments B+D As one skilled in the art will recognize, this single step method has utility in certain envisioned applications of the invention Figure 2 is a more detailed depiction of the recombinational cloning system of the invention, referred to herein as the "GATEWAY™ Cloning System " This figure depicts the production of Expression Clones via a "Destination Reaction, " which may also be referred to herein as an "LR Reaction " A kan r vector (referred to herein as an "Entry clone") containing a DNA molecule of interest (e.g., a gene) localized between an attLl site and an αttL2 site is reacted with an amp r vector (referred to herein as a "Destination Vector") containing a toxic or "death" gene localized between an crttRl site and an αttR2 site, in the presence of GATEWAY™ LR Clonase™ Enzyme Mix (a mixture of Int, IHF and Xis) After incubation at 25 °C for about 60 minutes, the reaction yields an amp r Expression Clone containing the DNA molecule of interest localized between an ttB 1 site and an αttB2 site, and a kan r byproduct molecule, as well as intermediates The reaction mixture may then be transformed into host cells (e.g. , E. coli) and clones containing the nucleic acid molecule of interest may be selected by plating the cells onto ampicillin-containing media and picking amp r colonies Figure 3 is a schematic depiction of the cloning of a nucleic acid molecule from an Entry clone into multiple types of Destination vectors, to produce a variety of Expression Clones Recombination between a given Entry clone and different types of Destination vectors (not shown), via the LR Reaction depicted in Figure 2, produces multiple different Expression Clones for use in a variety of applications and host cell types Figure 4 is a detailed depiction of the production of Entry Clones via a "BP reaction," also referred to herein as an "Entry Reaction" or a "Gateward Reaction " In the example shown in this figure, an amp r expression vector containing a DNA molecule of interest (e.g., a gene) localized between an αttBl site and an ttB2 site is reacted with a kan r Donor vector (e.g., an attP vector, here, GATEWAY™ pDONR201 (see Figure 49A-C)) containing a toxic or "death" gene localized between an αttPl site and an αttP2 site, in the presence of GATEWAY™ BP Clonase™ Enzyme Mix (a mixture of Int and IHF) After incubation at 25 °C for about 60 minutes, the reaction yields a kan r Entry clone containing the DNA molecule of interest localized between an αttLl site and an attL2 site, and an amp r by-product molecule The Entry clone may then be transformed into host cells (e.g., E. coli) and clones containing the Entry clone (and therefore the nucleic acid molecule of interest) may be selected by plating the cells onto kanamycin-containing media and picking kan r colonies Although this figure shows an example of use of a kan r Donor vector, it is also possible to use Donor vectors containing other selection markers, such as the gentamycin resistance or tetracycline resistance markers, as discussed herein Figure 5 is a more detailed schematic depiction of the LR ("Destination") reaction (Figure 5A) and the BP ("Entry" or "Gateward") reaction (Figure 5B) of the GATEWAY™ Cloning System, showing the reactants, products and byproducts of each reaction Figure 6 shows the sequences of the attB 1 and attB2 sites flanking a gene of interest after subcloning into a Destination Vector to create an Expression Clone Figure 7 is a schematic depiction of four ways to make Entry Clones using the compositions and methods of the invention 1 using restriction enzymes and ligase, 2 starting with a cDNA library prepared in an attL Entry Vector, 3 using an Expression Clone from a library prepared in an attB Expression Vector via the BxP reaction, and 4 recombinational cloning of PCR fragments with terminal attB sites, via the BxP reaction Approaches 3 and 4 rely on recombination with a Donor vector (here, an attP vector such as pDONR201 (see Figure 49A-C), pDONR202 (see Figure 50A-C), pDONR203 (see Figure 51A-C), pDONR204 (see Figure 52A-C), pDONR205 (see Figure 53A-C), or pDONR206 (see Figure 54A-C), for example) that provides an Entry Clone carrying a selection marker such as kan r , gen r , tet r , or the like Figure 8 is a schematic depiction of cloning of a PCR product by a BxP (Entry or Gateward) reaction A PCR product with 25 bp terminal attB sites (plus four Gs) is shown as a substrate for the BxP reaction Recombination between the attB-PCR product of a gene and a Donor vector (which donates an Entry Vector that carries kan r ) results in an Entry Clone of the PCR product Figure 9 is a listing of the nucleotide sequences of the recombination sites designated herein as αttBl, attB2, atiPl, αttP2, αttLl, αttL2, αttRl and αttR2 Sequences are written conventionally, from 5' to 3' Figures 10-20: The plasmid backbone for all the Entry Vectors depicted herein is the same, and is shown in Figure 10A for the Entry Vector pENTRIA For other Entry Vectors shown in Figures 11 -20, only the sequences shown in Figure "A" for each figure set (i.e., Figure 11 A, Figure 12 A, etc ) are different (within the attLl-attL2 cassettes) from those shown in Figure 10 — the plasmid backbone is identical Figure 10 is a schematic depiction of the physical map and cloning sites (Figure 10A), and the nucleotide sequence (Figure 10B), of the Entry Vector pENTRIA Figure 11 is a schematic depiction of the cloning sites (Figure 1 IA) and the nucleotide sequence (Figure 1 IB) of the Entry Vector pENTR2B Figure 12 is a schematic depiction of the cloning sites (Figure 12A) and the nucleotide sequence (Figure 12B) of the Entry Vector pENTR3C Figure 13 is a schematic depiction of the cloning sites (Figure 13 A) and the nucleotide sequence (Figure 13B) of the Entry Vector pENTR4 Figure 14 is a schematic depiction of the cloning sites (Figure 14A) and the nucleotide sequence (Figure 14B) of the Entry Vector pENTR5 Figure 15 is a schematic depiction of the cloning sites (Figure 15A) and the nucleotide sequence (Figure 15B) of the Entry Vector pENTR6 Figure 16 is a schematic depiction of the cloning sites (Figure 16 A) and the nucleotide sequence (Figure 16B) of the Entry Vector pENTR7 Figure 17 is a schematic depiction of the cloning sites (Figure 17 A) and the nucleotide sequence (Figure 17B) of the Entry Vector pENTR8 Figure 18 is a schematic depiction of the cloning sites (Figure 18 A) and the nucleotide sequence (Figure 18B) of the Entry Vector pENTR9 Figure 19 is a schematic depiction of the cloning sites (Figure 19A) and the nucleotide sequence (Figure 19B) of the Entry Vector pENTRlO Figure 20 is a schematic depiction of the cloning sites (Figure 20 A) and the nucleotide sequence (Figure 20B) of the Entry Vector pENTRl 1 Figure 21 is a schematic depiction of the physical map and the Trc expression cassette (Figure 21 A) showing the promoter sequences at -35 and at -10 from the initiation codon. and the nucleotide sequence (Figure 21B-D), of Destination Vector pDESTl This vector may also be referred to as pTrc- DEST1 Figure 22 is a schematic depiction of the physical map and the His6 expression cassette (Figure 22 A) showing the promoter sequences at -35 and at -10 from the initiation codon, and the nucleotide sequence (Figure 22B-D), of Destination Vector pDEST2 This vector may also be referred to as pHisό- DEST2 Figure 23 is a schematic depiction of the physical map and the GST expression cassette (Figure 23 A) showing the promoter sequences at -35 and at -10 from the initiation codon, and the nucleotide sequence (Figure 23B-D), of Destination Vector pDEST3 This vector may also be referred to as pGST- DEST3 Figure 24 is a schematic depiction of the physical map and the His6-Trx expression cassette (Figure 24 A) showing the promoter sequences at -35 and at - 10 from the initiation codon and a TEV protease cleavage site, and the nucleotide sequence (Figure 24B-D), of Destination Vector pDEST4 This vector may also be referred to as pTrx-DEST4 Figure 25 is a schematic depiction of the attRl and attR2 sites (Figure 25A), the physical map (Figure 25B), and the nucleotide sequence (Figure 25C-D), of Destination Vector pDEST5 This vector may also be referred to as pSPORT(+)-DEST5 Figure 26 is a schematic depiction of the attRl and attR2 sites (Figure 26A), the physical map (Figure 26B), and the nucleotide sequence (Figure 26C-D), of Destination Vector pDEST6 This vector may also be referred to as pSPORT(-)-DEST6 Figure 27 is a schematic depiction of the attRl site, CMV promoter, and the physical map (Figure 27 A), and the nucleotide sequence (Figure 27B-C), of Destination Vector pDEST7 This vector may also be referred to as pCMV- DEST7 Figure 28 is a schematic depiction of the attRl site, baculovirus polyhedrin promoter, and the physical map (Figure 28A), and the nucleotide sequence (Figure 28B-D), of Destination Vector pDEST8 This vector may also be referred to as pFastBac-DEST8 Figure 29 is a schematic depiction of the attRl site, Semliki Forest Virus promoter, and the physical map (Figure 29 A), and the nucleotide sequence (Figure 29B-E), of Destination Vector pDEST9 This vector may also be referred to as pSFV-DEST9 Figure 30 is a schematic depiction of the attRl site, baculovirus polyhedrin promoter, His6 fusion domain, and the physical map (Figure 30A), and the nucleotide sequence (Figure 30B-D), of Destination Vector pDESTIO This vector may also be referred to as pFastBacHT-DESTIO Figure 31 is a schematic depiction of the attRl cassette containing a tetracycline-regulated CMV promoter and the physical map (Figure 31 A), and the nucleotide sequence (Figure 31B-D), of Destination Vector pDESTl l This vector may also be referred to as pTet-DESTl 1 Figure 32 is a schematic depiction of the attRl site, the start of the mRNA of the CMV promoter, and the physical map (Figure 32A), and the nucleotide sequence (Figure 32B-D), of Destination Vector pDEST12 2 This vector may also be referred to as pCMVneo-DEST12, as pCMV-DEST12, or as pDEST12 Figure 33 is a schematic depiction of the attRl site, the λP L promoter, and the physical map (Figure 33 A), and the nucleotide sequence (Figure 33B-C), of Destination Vector pDEST13 This vector may also be referred to as pλP L - DEST13 Figure 34 is a schematic depiction of the attRl site, the T7 promoter, and the physical map (Figure 34A), and the nucleotide sequence (Figure 34B-D), of Destination Vector pDEST14 This vector may also be referred to as pPT7- DEST14 Figure 35 is a schematic depiction of the attRl site, the T7 promoter, and the N-terminal GST fusion sequence, and the physical map (Figure 35 A), and the nucleotide sequence (Figure 35B-D), of Destination Vector pDEST15 This vector may also be referred to as pT7 GST-DEST15 Figure 36 is a schematic depiction of the attRl site, the T7 promoter, and the N-terminal thioredoxin fusion sequence, and the physical map (Figure 36 A), and the nucleotide sequence (Figure 36B-D), of Destination Vector pDESTlό This vector may also be referred to as pT7 Trx-DEST16 Figure 37 is a schematic depiction of the attRl site, the T7 promoter, and the N-terminal His6 fusion sequence, and the physical map (Figure 37 A), and the nucleotide sequence (Figure 37B-D), of Destination Vector pDEST17. This vector may also be referred to as pT7 His-DEST17. Figure 38 is a schematic depiction of the attRl site and the plO baculovirus promoter, and the physical map (Figure 38 A), and the nucleotide sequence (Figure 38B-D), of Destination Vector pDEST 18. This vector may also be referred to as pFBpl0-DEST18. Figure 39 is a schematic depiction of the attRl site, and the 39k baculovirus promoter, and the physical map (Figure 39 A), and the nucleotide sequence (Figure 39B-D), of Destination Vector pDESTl 9. This vector may also be referred to as pFB39k-DEST19. Figure 40 is a schematic depiction of the attRl site, t cpolh baculovirus promoter, and the N-terminal GST fusion sequence, and the physical map (Figure 40A), and the nucleotide sequence (Figure 40B-D), of Destination Vector pDEST20. This vector may also be referred to as pFB GST-DEST20. Figure 41 is a schematic depiction of a 2-hybrid vector with a DNA- binding domain, the attRl site, and the ADH promoter, and the physical map (Figure 41 A), and the nucleotide sequence (Figure 41 B-E), of Destination Vector pDEST21. This vector may also be referred to as pDB Leu-DEST21. Figure 42 is a schematic depiction of a 2-hybrid vector with an activation domain, the attRl site, and the ADH promoter, and the physical map (Figure 42A), and the nucleotide sequence (Figure 42B-D), of Destination Vector pDEST22. This vector may also be referred to as pPC86-DEST22. Figure 43 is a schematic depiction of the attRl and attR2 sites, the T7 promoter, and the C-terminal His6 fusion sequence, and the physical map (Figure 43 A), and the nucleotide sequence (Figure 43B-D), of Destination Vector pDEST23. This vector may also be referred to as pC-term-His6-DEST23. Figure 44 is a schematic depiction of the attRl and attR2 sites, the T7 promoter, and the C-terminal GST fusion sequence, and the physical map (Figure 44A), and the nucleotide sequence (Figure 44B-D), of Destination Vector pDEST24. This vector may also be referred to as pC-term-GST-DEST24. Figure 45 is a schematic depiction of the attRl and attR2 sites, the T7 promoter, and the C-terminal thioredoxin fusion sequence, and the physical map (Figure 45 A), and the nucleotide sequence (Figure 45B-D), of Destination Vector pDEST25 This vector may also be referred to as pC-term-Trx-DEST25 Figure 46 is a schematic depiction of the attRl site, the CMV promoter, and an N-terminal His6 fusion sequence, and the physical map (Figure 46A), and the nucleotide sequence (Figure 46B-D), of Destination Vector pDEST26 This vector may also be referred to as pCMV-SPneo-His-DEST26 Figure 47 is a schematic depiction of the attRl site, the CMV promoter, and an N-terminal GST fusion sequence, and the physical map (Figure 47A), and the nucleotide sequence (Figure 47B-D), of Destination Vector pDEST27 This vector may also be referred to as pCMV-Spneo-GST-DEST27 Figure 48 is a depiction of the physical map (Figure 48 A), the cloning sites (Figure 48B), and the nucleotide sequence (Figure 48C-D), for the attB cloning vector plasmid pEXP501 This vector may also be referred to equivalently herein as pCMV'SPORTό, pCMVSPORTό, and pCMVSportό Figure 49 is a depiction of the physical map (Figure 49 A), and the nucleotide sequence (Figure 49B-C), for the Donor plasmid pDONR201 which donates a kanamycin-resistant vector in the BP Reaction This vector may also be referred to as pAttPkanr Donor Plasmid, or as pAttPkan Donor Plasmid Figure 50 is a depiction of the physical map (Figure 50A), and the nucleotide sequence (Figure 50B-C), for the Donor plasmid pDONR202 which donates a kanamycin-resistant vector in the BP Reaction Figure 51 is a depiction of the physical map (Figure 51 A), and the nucleotide sequence (Figure 51B-C), for the Donor plasmid pDONR203 which donates a kanamycin-resistant vector in the BP Reaction Figure 52 is a depiction of the physical map (Figure 52A), and the nucleotide sequence (Figure 52B-C), for the Donor plasmid pDONR204 which donates a kanamycin-resistant vector in the BP Reaction Figure 53 is a depiction of the physical map (Figure 53A), and the nucleotide sequence (Figure 53B-C), for the Donor plasmid pDONR205 which donates a tetracycline-resistant vector in the BP Reaction Figure 54 is a depiction of the physical map (Figure 54A), and the nucleotide sequence (Figure 54B-C), for the Donor plasmid pDONR206 which donates a gentamycin-resistant vector in the BP Reaction This vector may also be referred to as pENTR22 attP Donor Plasmid, pAttPGenr Donor Plasmid, or pAttPgent Donor Plasmid Figure 55 depicts the attBl site, and the physical map, of an Entry Clone (pENTR7) of CAT subcloned into the Destination Vector pDEST2 (Figure 22) Figure 56 depicts the DNA components of Reaction B of the one-tube BxP reaction described in Example 16, pEZC7102 and attB-tet-PCR Figure 57 is a physical map of the desired product of Reaction B of the one-tube BxP reaction described in Example 16, tetx7102 Figure 58 is a physical map of the Destination Vector pEZC8402 Figure 59 is a physical map of the expected te subclone product, tetx8402, resulting from the LxR Reaction with tetx7102 (Figure 57) plus pEZC8402 (Figure 58) Figure 60 is a schematic depiction of the bacteriophage lambda recombination pathways in E. coli Figure 61 is a schematic depiction of the DNA molecules participating in the LR Reaction Two different co-integrates form during the LR Reaction (only one of which is shown here), depending on whether attLl and attRl or attL2 and attR2 are first to recombine In one aspect, the invention provides directional cloning of a nucleic acid molecule of interest, since the recombination sites react with specificity (attLl reacts with attRl, attL2 with attR2, attBl with attPl, and attB2 with attP2) Thus, positioning of the sites allows construction of desired vectors having recombined fragments in the desired orientation Figure 62 is a depiction of native and fusion protein expression using the recombinational cloning methods and compositions of the invention In the upper figure depicting native protein expression, all of the translational start signals are included between the attBl and attB2 sites, therefore, these signals must be present in the starting Entry Clone The lower figure depicts fusion protein expression (here showing expression with both N-terminal and C-terminal fusion tags so that ribosomes read through attB 1 and attB2 to create the fusion protein) Unlike native protein expression vectors, N-terminal fusion vectors have their translational start signals upstream of the attBl site Figure 63 is a schematic depiction of three GATEWAY™ Cloning System cassettes Three blunt-ended cassettes are depicted which convert standard expression vectors to Destination Vectors Each of the depicted cassettes provides amino-terminal fusions in one of three possible reading frames, and each has a distinctive restriction cleavage site as shown Figure 64 shows the physical maps of plasmids containing three attR reading frame cassettes, pEZC 15101 (reading frame A, Figure 64 A), pEZC 15102 (reading frame B, Figure 64B), and pEZC15103 (reading frame C, Figure 64C) Figure 65 depicts the attB primers used for amplifying the tet r and amp r genes from pBR322 by the cloning methods of the invention Figure 66 is a table listing the results of recombinational cloning of the tet r and amp r PCR products made using the primers shown in Figure 65 Figure 67 is a graph showing the effect of the number of guanines (G's) contained on the 5' end of the PCR primers on the cloning efficiency of PCR products It is noted, however, that other nucleotides besides guanine (including A, T, C, U or combinations thereof) may be used as 5' extensions on the PCR primers to enhance cloning efficiency of PCR products Figure 68 is a graph showing a titration of various amounts of attP and attB reactants in the BxP reaction, and the effects on cloning efficiency of PCR products Figure 69 is a series of graphs showing the effects of various weights (Figure 69A) or moles (Figure 69B) of a 256 bp PCR product on formation of colonies, and on efficiency of cloning of the 256 bp PCR product into a Donor Vector (Figure 69C) Figure 70 is a series of graphs showing the effects of various weights (Figure 70A) or moles (Figure 70B) of a 1 kb PCR product on formation of colonies, and on efficiency of cloning of the 1 kb PCR product into a Donor Vector (Figure 70C) Figure 71 is a series of graphs showing the effects of various weights (Figure 71 A) or moles (Figure 7 IB) of a 1 4 kb PCR product on formation of colonies, and on efficiency of cloning of the 1 4 kb PCR product into a Donor Vector (Figure 71C) Figure 72 is a series of graphs showing the effects of various weights (Figure 72A) or moles (Figure 72B) of a 3 4 kb PCR product on formation of colonies, and on efficiency of cloning of the 3 4 kb PCR product into a Donor Vector (Figure 72C) Figure 73 is a series of graphs showing the effects of various weights (Figure 73 A) or moles (Figure 73B) of a 4 6 kb PCR product on formation of colonies, and on efficiency of cloning of the 4 6 kb PCR product into a Donor Vector (Figure 73 C) Figure 74 is photograph of an ethidium bromide- stained gel of a titration of a 6 9 kb PCR product in a BxP reaction Figure 75 is a graph showing the effects of various amounts of a 10 1 kb PCR product on formation of colonies upon cloning of the 10 1 kb PCR product into a Donor Vector Figure 76 is photograph of an ethidium bromide-stained gel of a titration of a 10 1 kb PCR product in a BxP reaction Figure 77 is a table summarizing the results of the PCR product cloning efficiency experiments depicted in Figures 69-74, for PCR fragments ranging in size from 0 256 kb to 6 9 kb Figure 78 is a depiction of the sequences at the ends of attR Cassettes Sequences contributed by the Cm r -ccdB cassette are shown, including the outer ends of the flanking attR sites (boxed) The staggered cleavage sites for Int are indicated in the boxed regions Following recombination with an Entry Clone, only the outer sequences in attR sites contribute to the resulting attB sites in the Expression Clone The underlined sequences at both ends dictate the different reading frames (reading frames A, B, or C, with two alternative reading frame C cassettes depicted) for fusion proteins Figure 79 is a depiction of several different attR cassettes (in reading frames A, B, or C) which may provide fusion codons at the amino-terminus of the encoded protein Figure 80 illustrates the single-cutting restriction sites in an attR reading frame A cassette of the invention Figure 81 illustrates the single-cutting restriction sites in an attR reading frame B cassette of the invention Figure 82 illustrates the single-cutting restriction sites in two alternative attR reading frame C cassettes of the invention (Figures 82 A and 82B) depicted in Figure 78 Figure 83 shows the physical map (Figure 83A), and the nucleotide sequence (Figure 83B-C), for an attR reading frame C parent plasmid prfC Parent III, which contains an attR reading frame C cassette of the invention (alternative A in Figures 78 and 82) Figure 84 is a physical map of plasmid pEZC1301 Figure 85 is a physical map of plasmid pEZC1313 Figure 86 is a physical map of plasmid pEZ14032 Figure 87 is a physical map of plasmid pMAB58 Figure 88 is a physical map of plasmid pMAB62 Figure 89 is a depiction of a synthesis reaction using two pairs of homologous primers of the invention Figure 90 is a schematic depiction of the physical map (Figure 90A), and the nucleotide sequence (Figure 90B-D), of Destination Vector pDEST28 Figure 91 is a schematic depiction of the physical map (Figure 91 A), and the nucleotide sequence (Figure 91B-D), of Destination Vector pDEST29 Figure 92 is a schematic depiction of the physical map (Figure 92A), and the nucleotide sequence (Figure 92B-D), of Destination Vector pDEST30 Figure 93 is a schematic depiction of the physical map (Figure 93 A), and the nucleotide sequence (Figure 93B-D), of Destination Vector pDEST31 Figure 94 is a schematic depiction of the physical map (Figure 94 A), and the nucleotide sequence (Figure 94B-E), of Destination Vector pDEST32 Figure 95 is a schematic depiction of the physical map (Figure 95A), and the nucleotide sequence (Figure 95B-D), of Destination Vector pDEST33 Figure 96 is a schematic depiction of the physical map (Figure 96A), and the nucleotide sequence (Figure 96B-D), of Destination Vector pDEST34 Figure 97 is a depiction of the physical map (Figure 97A), and the nucleotide sequence (Figure 97B-C), for the Donor plasmid pDONR207 which donates a gentamycin-resistant vector in the BP Reaction Figure 98 is a schematic depiction of the physical map (Figure 98 A), and the nucleotide sequence (Figure 98B-D), of the 2-hybrid vector pMAB85 Figure 99 is a schematic depiction of the physical map (Figure 99A), and the nucleotide sequence (Figure 99B-D), of the 2-hybrid vector pMAB86 DETAILED DESCRIPTION OF THE INVENTION Definitions In the description that follows, a number of terms used in recombinant DNA technology are utilized extensively In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided Byproduct is a daughter molecule (a new clone produced after the second recombination event during the recombinational cloning process) lacking the segment which is desired to be cloned or subcloned Cointegrate is at least one recombination intermediate nucleic acid molecule of the present invention that contains both parental (starting) molecules It will usually be linear In some embodiments it can be circular RNA and polypeptides may be expressed from cointegrates using an appropriate host cell strain, for example E. colιOB3 1 (particularly E. coli LIBRARY EFFICIENCY® DB3 1™ Competent Cells), and selecting for both selection markers found on the cointegrate molecule Host is any prokaryotic or eukaryotic organism that can be a recipient of the recombinational cloning Product, vector, or nucleic acid molecule of the invention A "host, " as the term is used herein, includes prokaryotic or eukaryotic organisms that can be genetically engineered For examples of such hosts, see Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982) Insert or Inserts include the desired nucleic acid segment or a population of nucleic acid segments (segment A of Figure 1) which may be manipulated by the methods of the present invention Thus, the terms Insert(s) are meant to include a particular nucleic acid (preferably DNA) segment or a population of segments Such Insert(s) can comprise one or more nucleic acid molecules Insert Donor is one of the two parental nucleic acid molecules (e g RNA or DNA) of the present invention which carries the Insert The Insert Donor molecule comprises the Insert flanked on both sides with recombination sites The Insert Donor can be linear or circular In one embodiment of the invention, the Insert Donor is a circular DNA molecule and further comprises a cloning vector sequence outside of the recombination signals (see Figure 1 ) When a population of Inserts or population of nucleic acid segments are used to make the Insert Donor, a population of Insert Donors results and may be used in accordance with the invention Examples of such Insert Donor molecules are GATEWAY™ Entry Vectors, which include but are not limited to those Entry Vectors depicted in Figures 10-20, as well as other vectors comprising a gene of interest flanked by one or more αttL sites (e.g. , attLl, attL2, etc ), or by one or more αttB sites (e.g. , ttBl, αttB2, etc ) for the production of library clones Product is one of the desired daughter molecules comprising the A and D sequences which is produced after the second recombination event during the recombinational cloning process (see Figure 1 ) The Product contains the nucleic acid which was to be cloned or subcloned In accordance with the invention, when a population of Insert Donors are used, the resulting population of Product molecules will contain all or a portion of the population of Inserts of the Insert Donors and preferably will contain a representative population of the original molecules of the Insert Donors Promoter is a DNA sequence generally described as the 5'-region of a gene, located proximal to the start codon The transcription of an adjacent DNA segment is initiated at the promoter region A repressible promoter's rate of transcription decreases in response to a repressing agent An inducible promoter's rate of transcription increases in response to an inducing agent A constitutive promoter's rate of transcription is not specifically regulated, though it can vary under the influence of general metabolic conditions Recognition sequence Recognition sequences are particular sequences which a protein, chemical compound, DNA, or RNA molecule (e.g., restriction endonuclease, a modification methylase, or a recombinase) recognizes and binds In the present invention, a recognition sequence will usually refer to a recombination site For example, the recognition sequence for Cre recombinase is loxP which is a 34 base pair sequence comprised of two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence See Figure 1 of Sauer, B , Current Opinion in Biotechnology 5 521-527 (1994) Other examples of recognition sequences are the attB, attP, attL, and attR sequences which are recognized by the recombinase enzyme A Integrase attB is an approximately 25 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region attP is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis) See Landy, Current Opinion in Biotechnology 3 699-707 (1993) Such sites may also be engineered according to the present invention to enhance production of products in the methods of the invention When such engineered sites lack the PI or HI domains to make the recombination reactions irreversible (e.g., attR or attP), such sites may be designated attR' or attP' to show that the domains of these sites have been modified in some way Recombination proteins include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites, which may be wild-type proteins (See Landy, Current Opinion in Biotechnology 3 699-707 (1993)), or mutants, derivatives (e g , fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof Recombination site is a recognition sequence on a DNA molecule participating in an integration/recombination reaction by the recombinational cloning methods of the invention Recombination sites are discrete sections or segments of DNA on the participating nucleic acid molecules that are recognized and bound by a site-specific recombination protein during the initial stages of integration or recombination For example, the recombination site for Cre recombinase is loxP which is a 34 base pair sequence comprised of two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence See Figure 1 of Sauer, B , Curr. Opm. Biotech. 5 521-527 ( 1994) Other examples of recognition sequences include the attB, attP, attL, and attR sequences described herein, and mutants, fragments, variants and derivatives thereof, which are recognized by the recombination protein A Int and by the auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis) See Landy, Curr. Opm. Biotech. 3 699-707 (1993) Recombinational Cloning is a method described herein, whereby segments of nucleic acid molecules or populations of such molecules are exchanged, inserted, replaced, substituted or modified, in vitro or in vivo By "in vitro" and "in vivo" herein is meant recombinational cloning that is carried out outside of host cells (e.g., in cell-free systems) or inside of host cells (e.g., using recombination proteins expressed by host cells), respectively Repression cassette is a nucleic acid segment that contains a repressor or a Selectable marker present in the subcloning vector Selectable marker is a DNA segment that allows one to select for or against a molecule (e.g. , a replicon) or a cell that contains it, often under particular conditions These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNN peptides, proteins, inorganic and organic compounds or compositions and the like Examples of Selectable markers include but are not limited to ( 1 ) DΝA segments that encode products which provide resistance against otherwise toxic compounds (e.g. , antibiotics), (2) DΝA segments that encode products which are otherwise lacking in the recipient cell (e.g., tRΝA genes, auxotrophic markers), (3) DΝA segments that encode products which suppress the activity of a gene product, (4) DΝA segments that encode products which can be readily identified (e.g. , phenotypic markers such as β-galactosidase, green fluorescent protein (GFP), and cell surface proteins), (5) DΝA segments that bind products which are otherwise detrimental to cell survival and/or function, (6) DΝA segments that otherwise inhibit the activity of any of the DΝA segments described in Νos 1 -5 above (e.g. , antisense oligonucleotides), (7) DΝA segments that bind products that modify a substrate (e.g. restriction endonucleases), (8) DΝA segments that can be used to isolate or identify a desired molecule (e.g. specific protein binding sites), (9) DΝA segments that encode a specific nucleotide sequence which can be otherwise nonfunctional (e.g. , for PCR amplification of subpopulations of molecules), ( 10) DΝA segments, which when absent, directly or indirectly confer resistance or sensitivity to particular compounds, (1 1) DΝA segments that encode products which are toxic in recipient cells, (12) DΝA segments that inhibit replication, partition or heritability of nucleic acid molecules that contain them, and/or (13) DΝA segments that encode conditional replication functions, e.g., replication in certain hosts or host cell strains or under certain environmental conditions (e.g. , temperature, nutritional conditions, etc ) Selection scheme is any method which allows selection, enrichment, or identification of a desired Product or Product(s) from a mixture containing an Entry Clone or Vector, a Destination Vector, a Donor Vector, an Expression Clone or Vector, any intermediates (e.g. a Cointegrate or a replicon), and/or Byproducts The selection schemes of one preferred embodiment have at least two components that are either linked or unlinked during recombinational cloning One component is a Selectable marker The other component controls the expression in vitro or in vivo of the Selectable marker, or survival of the cell (or the nucleic acid molecule, e.g. , a replicon) harboring the plasmid carrying the Selectable marker Generally, this controlling element will be a repressor or inducer of the Selectable marker, but other means for controlling expression or activity of the Selectable marker can be used Whether a repressor or activator is used will depend on whether the marker is for a positive or negative selection, and the exact arrangement of the various DNA segments, as will be readily apparent to those skilled in the art A preferred requirement is that the selection scheme results in selection of or enrichment for only one or more desired Products As defined herein, selecting for a DNA molecule includes (a) selecting or enriching for the presence of the desired DNA molecule, and (b) selecting or enriching against the presence of DNA molecules that are not the desired DNA molecule In one embodiment, the selection schemes (which can be carried out in reverse) will take one of three forms, which will be discussed in terms of Figure 1 The first, exemplified herein with a Selectable marker and a repressor therefore, selects for molecules having segment D and lacking segment C The second selects against molecules having segment C and for molecules having segment D Possible embodiments of the second form would have a DNA segment carrying a gene toxic to cells into which the in vitro reaction products are to be introduced A toxic gene can be a DNA that is expressed as a toxic gene product (a toxic protein or RNA), or can be toxic in and of itself (In the latter case, the toxic gene is understood to carry its classical definition of "heritable trait" ) Examples of such toxic gene products are well known in the art, and include, but are not limited to, restriction endonucleases (e.g., Dpnϊ), apoptosis- related genes (e g ASK1 or members of the bcl-2/ced-9 family), retroviral genes including those of the human immunodeficiency virus (HIV), defensins such as NP-1, inverted repeats or paired palindromic DNA sequences, bacteriophage lytic genes such as those from ΦX174 or bacteriophage T4, antibiotic sensitivity genes such as rpsL, antimicrobial sensitivity genes such aspheS, plasmid killer genes, eukaryotic transcriptional vector genes that produce a gene product toxic to bacteria, such as GATA-1, and genes that kill hosts in the absence of a suppressing function, e.g., kicB, ccdB, ΦX174 E (Liu, Q et αl., Curr. Biol 8 1300-1309 (1998)), and other genes that negatively affect replicon stability and/or replication A toxic gene can alternatively be selectable in vitro, e.g., a restriction site Many genes coding for restriction endonucleases operably linked to inducible promoters are known, and may be used in the present invention See, e.g. U S Patent Nos 4,960,707 (Dpnl and Dpnlϊ), 5,000,333, 5,082,784 and 5, 192,675 (Kpnl), 5, 141, %00 (NgoAlll and NgoAl), 5,179,015 (Fspl and Hαelll) 5,200,333 (Hαell and Tαql), 5,248,605 (Hpαll), 5,312,746 (CM), 5,231,021 and 5,304,480 (Xhol and Xholl), 5,334,526 (AM), 5,470,740 (Nsil), 5,534,428 (Sstl/SαcT), 5,202,248 (Ncol), 5, 139,942 (Ndel), and 5,098,839 (Pad) See also Wilson, G G , Nucl. Acids Res. 19 2539-2566 (1991), and Lunnen, K D , et al, Gene 74 25-32 (1988) In the second form, segment D carries a S electable marker The toxic gene would eliminate transformants harboring the Vector Donor, Cointegrate, and Byproduct molecules, while the Selectable marker can be used to select for cells containing the Product and against cells harboring only the Insert Donor The third form selects for cells that have both segments A and D in cis on the same molecule, but not for cells that have both segments in trans on different molecules This could be embodied by a Selectable marker that is split into two inactive fragments, one each on segments A and D The fragments are so arranged relative to the recombination sites that when the segments are brought together by the recombination event, they reconstitute a functional Selectable marker For example, the recombinational event can link a promoter with a structural nucleic acid molecule (e.g., a gene), can link two fragments of a structural nucleic acid molecule, or can link nucleic acid molecules that encode a heterodimeric gene product needed for survival, or can link portions of a replicon Site-specific recombinase is a type of recombinase which typically has at least the following four activities (or combinations thereof) ( 1 ) recognition of one or two specific nucleic acid sequences, (2) cleavage of said sequence or sequences, (3) topoisomerase activity involved in strand exchange, and (4) ligase activity to reseal the cleaved strands of nucleic acid See Sauer, B , Current Opinions in Biotechnology 5 521-527 (1994) Conservative site-specific recombination is distinguished from homologous recombination and transposition by a high degree of sequence specificity for both partners The strand exchange mechanism involves the cleavage and rejoining of specific DNA sequences in the absence of DNA synthesis (Landy, A (1989) Ann. Rev. Biochem. 58 913-949) Subcloning vector is a cloning vector comprising a circular or linear nucleic acid molecule which includes preferably an appropriate replicon In the present invention, the subcloning vector (segment D in Figure 1) can also contain functional and/or regulatory elements that are desired to be incorporated into the final product to act upon or with the cloned DNA Insert (segment^ in Figure 1) The subcloning vector can also contain a Selectable marker (preferably DNA) Vector is a nucleic acid molecule (preferably DNA) that provides a useful biological or biochemical property to an Insert Examples include plasmids, phages, autonomously replicating sequences (ARS), centromeres, and other sequences which are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell A Vector can have one or more restriction endonuclease recognition sites at which the sequences can be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a nucleic acid fragment can be spliced in order to bring about its replication and cloning Vectors can further provide primer sites, e.g., for PCR, transcriptional and/or translational initiation and/or regulation sites, recombinational signals, replicons, Selectable markers, etc Clearly, methods of inserting a desired nucleic acid fragment which do not require the use of homologous recombination, transpositions or restriction enzymes (such as, but not limited to, UDG cloning of PCR fragments (U S Patent No 5,334,575, entirely incorporated herein by reference), T A cloning, and the like) can also be applied to clone a fragment into a cloning vector to be used according to the present invention The cloning vector can further contain one or more selectable markers suitable for use in the identification of cells transformed with the cloning vector Vector Donor is one of the two parental nucleic acid molecules (e g RNA or DNA) of the present invention which carries the DNA segments comprising the DNA vector which is to become part of the desired Product The Vector Donor comprises a subcloning vector D (or it can be called the cloning vector if the Insert Donor does not already contain a cloning vector (e.g. , for PCR fragments containing αttB sites, see below)) and a segment C flanked by recombination sites (see Figure 1) Segments C and/or D can contain elements that contribute to selection for the desired Product daughter molecule, as described above for selection schemes The recombination signals can be the same or different, and can be acted upon by the same or different recombinases In addition, the Vector Donor can be linear or circular Examples of such Vector Donor molecules include GATEWAY™ Destination Vectors, which include but are not limited to those Destination Vectors depicted in Figures 21-47 and 90-96 Primer refers to a single stranded or double stranded oligonucleotide that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule (e g a DNA molecule) In a preferred aspect, a primer comprises one or more recombination sites or portions of such recombination sites Portions of recombination sites comprise at least 2 bases (or basepairs, abbreviated herein as "bp"), at least 5-200 bases, at least 10-100 bases, at least 15-75 bases, at least 15-50 bases, at least 15-25 bases, or at least 16-25 bases, of the recombination sites of interest, as described in further detail below and in the Examples When using portions of recombination sites, the missing portion of the recombination site may be provided as a template by the newly synthesized nucleic acid molecule Such recombination sites may be located within and/or at one or both termini of the primer Preferably, additional sequences are added to the primer adjacent to the recombination site(s) to enhance or improve recombination and/or to stabilize the recombination site during recombination Such stabilization sequences may be any sequences (preferably G/C rich sequences) of any length Preferably, such sequences range in size from 1 to about 1000 bases, 1 to about 500 bases, and 1 to about 100 bases, 1 to about 60 bases, 1 to about 25, 1 to about 10, 2 to about 10 and preferably about 4 bases Preferably, such sequences are greater than 1 base in length and preferably greater than 2 bases in length Template refers to double stranded or single stranded nucleic acid molecules which are to be amplified, synthesized or sequenced In the case of double stranded molecules, denaturation of its strands to form a first and a second strand is preferably performed before these molecules will be amplified, synthesized or sequenced, or the double stranded molecule may be used directly as a template For single stranded templates, a primer complementary to a portion of the template is hybridized under appropriate conditions and one or more polypeptides having polymerase activity (e g DNA polymerases and/or reverse transcriptases) may then synthesize a nucleic acid molecule complementary to all or a portion of said template Alternatively, for double stranded templates, one or more promoters may be used in combination with one or more polymerases to make nucleic acid molecules complementary to all or a portion of the template The newly synthesized molecules, according to the invention, may be equal or shorter in length than the original template Additionally, a population of nucleic acid templates may be used during synthesis or amplification to produce a population of nucleic acid molecules typically representative of the original template population Adapter is an oligonucleotide or nucleic acid fragment or segment (preferably DNA) which comprises one or more recombination sites (or portions of such recombination sites) which in accordance with the invention can be added to a circular or linear Insert Donor molecule as well as other nucleic acid molecules described herein When using portions of recombination sites, the missing portion may be provided by the Insert Donor molecule Such adapters may be added at any location within a circular or linear molecule, although the adapters are preferably added at or near one or both termini of a linear molecule Preferably, adapters are positioned to be located on both sides (flanking) a particular nucleic acid molecule of interest In accordance with the invention, adapters may be added to nucleic acid molecules of interest by standard recombinant techniques (e g restriction digest and ligation) For example, adapters may be added to a circular molecule by first digesting the molecule with an appropriate restriction enzyme, adding the adapter at the cleavage site and reforming the circular molecule which contains the adapter(s) at the site of cleavage In other aspects, adapters may be added by homologous recombination, by integration of RNA molecules, and the like Alternatively, adapters may be ligated directly to one or more and preferably both termini of a linear molecule thereby resulting in linear molecule(s) having adapters at one or both termini In one aspect of the invention, adapters may be added to a population of linear molecules, (e g a cDNA library or genomic DNA which has been cleaved or digested) to form a population of linear molecules containing adapters at one and preferably both termini of all or substantial portion of said population Adapter-Primer is primer molecule which comprises one or more recombination sites (or portions of such recombination sites) which in accordance with the invention can be added to a circular or linear nucleic acid molecule described herein When using portions of recombination sites, the missing portion may be provided by a nucleic acid molecule (e.g., an adapter) of the invention Such adapter-primers may be added at any location within a circular or linear molecule, although the adapter-primers are preferably added at or near one or both termini of a linear molecule Examples of such adapter-primers and the use thereof in accordance with the methods of the invention are shown in Example 25 herein Such adapter-primers may be used to add one or more recombination sites or portions thereof to circular or linear nucleic acid molecules in a variety of contexts and by a variety of techniques, including but not limited to amplification (e.g., PCR), ligation (e.g. , enzymatic or chemical/synthetic ligation), recombination (e.g. , homologous or non-homologous (illegitimate) recombination) and the like Library refers to a collection of nucleic acid molecules (circular or linear) In one embodiment, a library may comprise a plurality (/. e. , two or more) of DNA molecules, which may or may not be from a common source organism, organ, tissue, or cell In another embodiment, a library is representative of all or a portion or a significant portion of the DNA content of an organism (a "genomic" library), or a set of nucleic acid molecules representative of all or a portion or a significant portion of the expressed nucleic acid molecules (a cDNA library) in a cell, tissue, organ or organism A library may also comprise random sequences made by de novo synthesis, mutagenesis of one or more sequences and the like Such libraries may or may not be contained in one or more vectors Amplification refers to any in vitro method for increasing a number of copies of a nucleotide sequence with the use of a polymerase Nucleic acid amplification results in the incorporation of nucleotides into a DNA and/or RNA molecule or primer thereby forming a new molecule complementary to a template The formed nucleic acid molecule and its template can be used as templates to synthesize additional nucleic acid molecules As used herein, one amplification reaction may consist of many rounds of replication DNA amplification reactions include, for example, polymerase chain reaction (PCR) One PCR reaction may consist of 5-100 "cycles" of denaturation and synthesis of a DNA molecule Oligonucleotide refers to a synthetic or natural molecule comprising a covalently linked sequence of nucleotides which are joined by a phosphodiester bond between the 3' position of the deoxyribose or ribose of one nucleotide and the 5' position of the deoxyribose or ribose of the adjacent nucleotide This term may be used interchangeably herein with the terms "nucleic acid molecule" and "polynucleotide," without any of these terms necessarily indicating any particular length of the nucleic acid molecule to which the term specifically refers Nucleotide refers to a base-sugar-phosphate combination Nucleotides are monomeric units of a nucleic acid molecule (DNA and RNA) The term nucleotide includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof Such derivatives include, for example, [αSjdATP, 7-deaza-dGTP and 7-deaza-dATP The term nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives Illustrated examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddlTP, and ddTTP According to the present invention, a "nucleotide" may be unlabeled or detectably labeled by well known techniques Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels Hybridization The terms "hybridization" and "hybridizing" refers to base pairing of two complementary single-stranded nucleic acid molecules (RNA and/or DNA) to give a double stranded molecule As used herein, two nucleic acid molecules may be hybridized, although the base pairing is not completely complementary Accordingly, mismatched bases do not prevent hybridization of two nucleic acid molecules provided that appropriate conditions, well known in the art, are used In some aspects, hybridization is said to be under "stringent conditions " By "stringent conditions" as used herein is meant overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0 lx SSC at about 65 °C Other terms used in the fields of recombinant DNA technology and molecular and cell biology as used herein will be generally understood by one of ordinary skill in the applicable arts Overview Two reactions constitute the recombinational cloning system of the present invention, referred to herein as the "GATEWAY™ Cloning System," as depicted generally in Figure 1 The first of these reactions, the LR Reaction (Figure 2), which may also be referred to interchangeably herein as the Destination Reaction, is the main pathway of this system The LR Reaction is a recombination reaction between an Entry vector or clone and a Destination Vector, mediated by a cocktail of recombination proteins such as the GATEWAY™ LR Clonase™ Enzyme Mix described herein This reaction transfers nucleic acid molecules of interest (which may be genes, cDNAs, cDNA libraries, or fragments thereof) from the Entry Clone to an Expression Vector, to create an Expression Clone The sites labeled L, R, B , and P are respectively the attL, attR, attB, and attP recombination sites for the bacteriophage A recombination proteins that constitute the Clonase cocktail (referred to herein variously as "Clonase" or "GATEWAY™ LR Clonase™ Enzyme Mix" (for recombination protein mixtures mediating attL x attR recombination reactions, as described herein) or "GATEWAY™ BP Clonase™ Enzyme Mix" (for recombination protein mixtures mediating attB x attP recombination reactions, as described herein)) The Recombinational Cloning reactions are equivalent to concerted, highly specific, cutting and ligation reactions Viewed in this way, the recombination proteins cut to the left and right of the nucleic acid molecule of interest in the Entry Clone and ligate it into the Destination vector, creating a new Expression Clone The nucleic acid molecule of interest in an Expression Clone is flanked by the small attBl and attB2 sites The orientation and reading frame of the nucleic acid molecule of interest are maintained throughout the subcloning, because attLl reacts only with attRl, and attL2 reacts only with attR2 Likewise, attBl reacts only with attP 1 , and attB2 reacts only with attP2 Thus, the invention also relates to methods of controlled or directional cloning using the recombination sites of the invention (or portions thereof), including variants, fragments, mutants and derivatives thereof which may have altered or enhanced specificity The invention also relates more generally to any number of recombination site partners or pairs (where each recombination site is specific for and interacts with its corresponding recombination site) Such recombination sites are preferably made by mutating or modifying the recombination site to provide any number of necessary specificities (e.g., attB 1-10, attPl-10, attLl-10, attRl -10, etc ), non-limiting examples of which are described in detail in the Examples herein When an aliquot from the recombination reaction is transformed into host cells (e.g. , E. coli) and spread on plates containing an appropriate selection agent, e.g. , an antibiotic such as ampicillin with or without methicillin, cells that take up the desired clone form colonies The unreacted Destination Vector does not give ampicillin-resistant colonies, even though it carries the ampicillin-resistance gene, because it contains a toxic gene, e.g., ccdB Thus selection for ampicillin resistance selects for E. coli cells that carry the desired product, which usually comprise >90% of the colonies on the ampicillin plate To participate in the Recombinational (or "GATEWAY™") Cloning Reaction, a nucleic acid molecule of interest first may be cloned into an Entry Vector, creating an Entry Clone Multiple options are available for creating Entry Clones, including cloning of PCR sequences with terminal attB recombination sites into Entry Vectors, using the GATEWAY™ Cloning System recombination reaction, transfer of genes from libraries prepared in GATEWAY™ Cloning System vectors by recombination into Entry Vectors, and cloning of restriction enzyme- generated fragments and PCR fragments into Entry Vectors by standard recombinant DNA methods These approaches are discussed in further detail herein A key advantage of the GATEWAY™ Cloning System is that a nucleic acid molecule of interest (or even a population of nucleic acid molecules of interest) present as an Entry Clone can be subcloned in parallel into one or more Destination Vectors in a simple reactions for anywhere from about 30 seconds to about 60 minutes (preferably about 1-60 minutes, about 1-45 minutes, about 1-30 minutes, about 2-60 minutes, about 2-45 minutes, about 2-30 minutes, about 1-2 minutes, about 30-60 minutes, about 45-60 minutes, or about 30-45 minutes) Longer reaction times (e.g. , 2-24 hours, or overnight) may increase recombination efficiency, particularly where larger nucleic acid molecules are used, as described in the Examples herein Moreover, a high percentage of the colonies obtained carry the desired Expression Clone This process is illustrated schematically in Figure 3, which shows an advantage of the invention in which the molecule of interest can be moved simultaneously or separately into multiple Destination Vectors In the LR Reaction, one or both of the nucleic acid molecules to be recombined may have any topology (e.g., linear, relaxed circular, nicked circular, supercoiled, etc ), although one or both are preferably linear The second major pathway of the GATEWAY™ Cloning System is the BP Reaction (Figure 4), which may also be referred to interchangeably herein as the Entry Reaction or the Gateward Reaction The BP Reaction may recombine an Expression Clone with a Donor Plasmid (the counterpart of the byproduct in Figure 2) This reaction transfers the nucleic acid molecule of interest (which may have any of a variety of topologies, including linear, coiled, supercoiled, etc ) in the Expression Clone into an Entry Vector, to produce a new Entry Clone Once this nucleic acid molecule of interest is cloned into an Entry Vector, it can be transferred into new Expression Vectors, through the LR Reaction as described above In the BP Reaction, one or both of the nucleic acid molecules to be recombined may have any topology (e.g., linear, relaxed circular, nicked circular, supercoiled, etc ), although one or both are preferably linear A useful variation of the BP Reaction permits rapid cloning and expression of products of amplification (e.g., PCR) or nucleic acid synthesis Amplification (e.g. , PCR) products synthesized with primers containing terminal 25 bp attB sites serve as efficient substrates for the Gateward Cloning reaction Such amplification products may be recombined with a Donor Vector to produce an Entry Clone (see Figure 7) The result is an Entry Clone containing the amplification fragment Such Entry Clones can then be recombined with Destination Vectors ~ through the LR Reaction — to yield Expression Clones of the PCR product Additional details of the LR Reaction are shown in Figure 5A The GATEWAY™ LR Clonase™ Enzyme Mix that mediates this reaction contains lambda recombination proteins Int (Integrase), Xis (Excisionase), and IHF (Integration Host Factor) In contrast, the GATEWAY™ BP Clonase™ Enzyme Mix, which mediates the BP Reaction (Figure 5B), comprises Int and IHF alone The recombination (att) sites of each vector comprise two distinct segments, donated by the parental vectors The staggered lines dividing the two portions of each att site, depicted in Figures 5 A and 5B, represent the seven-base staggered cut produced by Int during the recombination reactions This structure is seen in greater detail in Figure 6, which displays the attB recombination sequences of an Expression Clone, generated by recombination between the attLl and attL2 sites of an Entry Clone and the attRl and attR2 sites of a Destination Vector The nucleic acid molecule of interest in the Expression Clone is flanked by attB sites attBl to the left (amino terminus) and attB2 to the right (carboxy terminus) The bases in attB 1 to the left of the seven-base staggered cut produced by Int are derived from the Destination vector, and the bases to the right of the staggered cut are derived from the Entry Vector (see Figure 6) Note that the sequence is displayed in triplets corresponding to an open reading frame If the reading frame of the nucleic acid molecule of interest cloned in the Entry Vector is in phase with the reading frame shown for attB 1 , amino-terminal protein fusions can be made between the nucleic acid molecule of interest and any GATEWAY™ Cloning System Destination Vector encoding an amino-terminal fusion domain Entry Vectors and Destination Vectors that enable cloning in all three reading frames are described in more detail herein, particularly in the Examples The LR Reaction allows the transfer of a desired nucleic acid molecule of interest into new Expression Vectors by recombining a Entry Clone with various Destination Vectors To participate in the LR or Destination Reaction, however, a nucleic acid molecule of interest preferably is first converted to a Entry Clone Entry Clones can be made in a number of ways, as shown in Figure 7 One approach is to clone the nucleic acid molecule of interest into one or more of the Entry Vectors, using standard recombinant DNA methods, with restriction enzymes and ligase The starting DNA fragment can be generated by restriction enzyme digestion or as a PCR product The fragment is cloned between the attLl and attL2 recombination sites in the Entry Vector Note that a toxic or "death" gene (e.g., ccdB), provided to minimize background colonies from incompletely digested Entry Vector, must be excised and replaced by the nucleic acid molecule of interest A second approach to making an Entry Clone (Figure 7) is to make a library (genomic or cDNA) in an Entry Vector, as described in detail herein Such libraries may then be transferred into Destination Vectors for expression screening, for example in appropriate host cells such as yeast cells or mammalian cells A third approach to making Entry Clones (Figure 7) is to use Expression Clones obtained from cDNA molecules or libraries prepared in Expression Vectors Such cDNAs or libraries, flanked by attB sites, can be introduced into a Entry Vector by recombination with a Donor Vector via the BP Reaction If desired, an entire Expression Clone library can be transferred into the Entry Vector through the BP Reaction Expression Clone cDNA libraries may also be constructed in a variety of prokaryotic and eukaryotic GATEWAY™-modified vectors (e.g., the pEXP501 Expression Vector (see Figure 48), and 2-hybrid and attB library vectors), as described in detail herein, particularly in the Examples below A fourth, and potentially most versatile, approach to making an Entry Clone (Figure 7) is to introduce a sequence for a nucleic acid molecule of interest into an Entry Vector by amplification (e.g. , PCR) fragment cloning This method is diagramed in Figure 8 The DNA sequence first is amplified (for example, with PCR) as outlined in detail below and in the Examples herein, using primers containing one or more bp, two or more bp, three or more bp, four or more bp, five or more bp, preferably six or more bp, more preferably 6-25 bp (particularly 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) bp of the attB nucleotide sequences (such as, but not limited to, those depicted in Figure 9), and optionally one or more, two or more, three or more, four or more, and most preferably four or five or more additional terminal nucleotide bases which preferably are guanines The PCR product then may be converted to a Entry Clone by performing a BP Reaction, in which the attB-PCR product recombines with a Donor Vector containing one or more attP sites Details of this approach and protocols for PCR fragment subcloning are provided in Examples 8 and 21-25 A variety of Entry Clones may be produced by these methods, providing a wide array of cloning options, a number of specific Entry Vectors are also available commercially from Life Technologies, Inc (Rockville, MD) The Examples herein provide a more in-depth description of selected Entry Vectors and details of their cloning sites Choosing the optimal Entry Vector for a particular application is discussed in Example 4 Entry Vectors and Destination Vectors should be constructed so that the amino-terminal region of a nucleic acid molecule of interest (e.g., a gene, cDNA library or insert, or fragment thereof) will be positioned next to the attLl site Entry Vectors preferably contain the rrnB transcriptional terminator upstream of the attLl site This sequence ensures that expression of cloned nucleic acid molecules of interest is reliably "off" in E. coli, so that even toxic genes can be successfully cloned Thus, Entry Clones may be designed to be transcriptionally silent Note also that Entry Vectors, and hence Entry Clones, may contain the kanamycin antibiotic resistance (kan r ) gene to facilitate selection of host cells containing Entry Clones after transformation In certain applications, however, Entry Clones may contain other selection markers, including but not limited to a gentamycin resistance (gen r ) or tetracycline resistance (tet r ) gene, to facilitate selection of host cells containing Entry Clones after transformation Once a nucleic acid molecule of interest has been cloned into an Entry Vector, it may be moved into a Destination Vector The upper right portion of Figure 5 A shows a schematic of a Destination Vector The thick arrow represents some function (often transcription or translation) that will act on the nucleic acid molecule of interest in the clone During the recombination reaction, the region between the attRl and attR2 sites, including a toxic or "death" gene (e.g. , ccdB), is replaced by the DNA segment from the Entry Clone Selection for recombinants that have acquired the ampicillin resistance (amp r ) gene (carried on the Destination Vector) and that have also lost the death gene ensures that a high percentage (usually >90%) of the resulting colonies will contain the correct insert To move a nucleic acid molecule of interest into a Destination Vector, the Destination Vector is mixed with the Entry Clone comprising the desired nucleic acid molecule of interest, a cocktail of recombination proteins (e.g. , GATEWAY™ LR Clonase™ Enzyme Mix) is added, the mixture is incubated (preferably at about 25 °C for about 60 minutes, or longer under certain circumstances, e.g. for transfer of large nucleic acid molecules, as described below) and any standard host cell (including bacterial cells such asE. col , animal cells such as insect cells, mammalian cells, nematode cells and the like; plant cells, and yeast cells) strain is transformed with the reaction mixture The host cell used will be determined by the desired selection (e.g., E. coli DB3 1, available commercially from Life Technologies, Inc., allows survival of clones containing the ccdB death gene, and thus can be used to select for cointegrate molecules — /. e. , molecules that are hybrids between the Entry Clone and Destination Vector) The Examples below provide further details and protocols for use of Entry and Destination Vectors in transferring nucleic acid molecules of interest and expressing RNAs or polypeptides encoded by these nucleic acid molecules in a variety of host cells The cloning system of the invention therefore offers multiple advantages • Once a nucleic acid molecule of interest is cloned into the GATEWAY™ Cloning System, it can be moved into and out of other vectors with complete fidelity of reading frame and orientation That is, since the reactions proceed whereby attLl on the Entry Clone recombines with attRl on the Destination Vector, the directionality of the nucleic acid molecule of interest is maintained or may be controlled upon transfer from the Entry Clone into the Destination Vector Hence, the GATEWAY™ Cloning System provides a powerful and easy method of directional cloning of nucleic acid molecule of interest • One-step cloning or subcloning Mix the Entry Clone and the Destination Vector with Clonase, incubate, and transform • Clone PCR products readily by in vitro recombination, by adding attB sites to PCR primers Then directly transfer these Entry Clones into Destination Vectors This process may also be carried out in one step (see Examples below) • Powerful selections give high reliability >90% ( and often >99%) of the colonies contain the desired DNA in its new vector • One-step conversion of existing standard vectors into GATEWAY™ Cloning System vectors • Ideal for large vectors or those with few cloning sites • Recombination sites are short (25 bp), and may be engineered to contain no stop codons or secondary structures • Reactions may be automated, for high-throughput applications (e.g., for diagnostic purposes or for therapeutic candidate screening) • The reactions are economical 0 3 μg of each DNA, no restriction enzymes, phosphatase, ligase, or gel purification Reactions work well with miniprep DNA • Transfer multiple clones, and even libraries, into one or more Destination Vectors, in a single experiment • A variety of Destination Vectors may be produced, for applications including, but not limited to •Protein expression in E. coli native proteins, fusion proteins with GST, Hisό, thioredoxin, etc , for purification, or one or more epitope tags, any promoter useful in expressing proteins in E. coli may be used, such as ptrc, AP L , and T7 promoters 'Protein expression in eukaryotic cells CMV promoter, baculovirus (with or without His6 tag), Semliki Forest virus, Tet regulation •DNA sequencing (all lac primers), RNA probes, phagemids (both strands) • A variety of Entry Vectors (for recombinational cloning entry by standard recombinant DNA methods) may be produced •Strong transcription stop just upstream, for genes toxic to E. coli. •Three reading frames •With or without TEV protease cleavage site •Motifs for prokaryotic and / or eukaryotic translation • Compatible with commercial cDNA libraries • Expression Clone cDNA (attB) libraries, for expression screening, including 2-hybrid libraries and phage display libraries, may also be constructed Recombination Site Sequences In one aspect, the invention relates to nucleic acid molecules, which may or may not be isolated nucleic acid molecules, comprising one or more nucleotide sequences encoding one or more recombination sites or portions thereof In particular, this aspect of the invention relates to such nucleic acid molecules comprising one or more nucleotide sequences encoding attB, attP, attL, or αttR, or portions of these recombination site sequences The invention also relates to mutants, derivatives, and fragments of such nucleic acid molecules Unless otherwise indicated, all nucleotide sequences that may have been determined by sequencing a DNA molecule herein were determined using manual or automated DNA sequencing, such as dideoxy sequencing, according to methods that are routine to one of ordinary skill in the art (Sanger, F , and Coulson, A R , J. Mol. Biol. 94 444-448 (1975), Sanger, F , et al, Proc. Natl. Acad. Sci. USA 74 5463- 5467 (1977)) All amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by conceptual translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by these approaches, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by such methods are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion. Unless otherwise indicated, each "nucleotide sequence" set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G , C and T). However, by "nucleotide sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C andU), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U). Thus, the invention relates to sequences of the invention in the form of DNA or RNA molecules, or hybrid DNA RNA molecules, and their corresponding complementary DNA, RNA, or DNA RNA strands. In a first such aspect, the invention provides nucleic acid molecules comprising one or more nucleotide sequences encoding ttB 1 , or mutants, fragments, variants or derivatives thereof. Such nucleic acid molecules may comprise an αttBl nucleotide sequence having the sequence set forth in Figure 9, such as: ACAAGTTTGTACAAAAAAGCAGGCT, or a nucleotide sequence complementary to the nucleotide sequence set forth in Figure 9 for αttBl, or mutants, fragments, variants or derivatives thereof. As one of ordinary skill will appreciate, however, certain mutations, insertions, or deletions of one or more bases in the ttB 1 sequence contained in the nucleic acid molecules of the invention may be made without compromising the structural and functional integrity of these molecules, hence, nucleic acid molecules comprising such mutations, insertions, or deletions in the αttB 1 sequence are encompassed within the scope of the invention In a related aspect, the invention provides nucleic acid molecules comprising one or more nucleotide sequences encoding αttB2, or mutants, fragments, variants or derivatives thereof Such nucleic acid molecules may comprise an αttB2 nucleotide sequence having the sequence set forth in Figure 9, such as ACCCAGCTTTCTTGTACAAAGTGGT, or a nucleotide sequence complementary to the nucleotide sequence set forth in Figure 9 for αttB2, or mutants, fragments, variants or derivatives thereof As noted above for αttBl, certain mutations, insertions, or deletions of one or more bases in the αttB2 sequence contained in the nucleic acid molecules of the invention may be made without compromising the structural and functional integrity of these molecules, hence, nucleic acid molecules comprising such mutations, insertions, or deletions in the αttB2 sequence are encompassed within the scope of the invention A recombinant host cell comprising a nucleic acid molecule containing attB 1 and attB2 sites (the vector pEXP501, also known as pCMNSportό, see Figure 48), E. coli DB3 l(pCMVSport6), was deposited on February 27, 1999, with the Collection, Agricultural Research Culture Collection (ΝRRL), 1815 North University Street, Peoria, Illinois 61604 USA, as Deposit No NRRL B- 30108 The attB 1 and attB2 sites within the deposited nucleic acid molecule are contained in nucleic acid cassettes in association with one or more additional functional sequences as described in more detail below In another related aspect, the invention provides nucleic acid molecules comprising one or more nucleotide sequences encoding αttPl, or mutants, fragments, variants or derivatives thereof Such nucleic acid molecules may comprise an αttP 1 nucleotide sequence having the sequence set forth in Figure 9, such as TACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGA- CTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTAT- GCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTT- TCTCGTTCAGCTTTTTTGTACAAAGTTGGCATTATAAAAAAGCATTG- CTCATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAA- ANTCNTTATTTG, or a nucleotide sequence complementary to the nucleotide sequence set forth in Figure 9 for ttPl, or mutants, fragments, variants or derivatives thereof. As noted above for αttB 1 , certain mutations, insertions, or deletions of one or more bases in the αttPl sequence contained in the nucleic acid molecules of the invention may be made without compromising the structural and functional integrity of these molecules; hence, nucleic acid molecules comprising such mutations, insertions, or deletions in the αttPl sequence are encompassed within the scope of the invention. In another related aspect, the invention provides nucleic acid molecules comprising one or more nucleotide sequences encoding αttP2, or mutants, fragments, variants or derivatives thereof. Such nucleic acid molecules may comprise an αttP2 nucleotide sequence having the sequence set forth in Figure 9, such as: CAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTG- CAACAAATTGATAAGCAATGCTTTCTTATAATGCCAACTTT- G T A C A A G A AA G C T G A A C G A G A A A C G T AAAA T GA T A - TAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAG- ACTACATANTACTGTANAACACAACATATCCAGTCACTATGAATCAA- CTACTTAGATGGTATTAGTGACCTGTN or a nucleotide sequence complementary to the nucleotide sequence set forth in Figure 9 for αttP2, or mutants, fragments, variants or derivatives thereof . As noted above for αttBl, certain mutations, insertions, or deletions of one or more bases in the αttP2 sequence contained in the nucleic acid molecules of the invention may be made without compromising the structural and functional integrity of these molecules; hence, nucleic acid molecules comprising such mutations, insertions, or deletions in the αttP2 sequence are encompassed within the scope of the invention. A recombinant host cell comprising a nucleic acid molecule (the attP vector pDOΝR201, also known as pENTR21-attPkan or pAttPkan; see Figure 49) containing attPl and attP2 sites, E. coli DB3.1(pAttPkan) (also called E. coli DB3.1(pAHKan)), was deposited on February 27, 1999, with the Collection, Agricultural Research Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA, as Deposit No. NRRL B-30099. The attPl and attP2 sites within the deposited nucleic acid molecule are contained in nucleic acid cassettes in association with one or more additional functional sequences as described in more detail below In another related aspect, the invention provides nucleic acid molecules comprising one or more nucleotide sequences encoding αttRl, or mutants, fragments, variants or derivatives thereof Such nucleic acid molecules may comprise an αttRl nucleotide sequence having the sequence set forth in Figure 9, such as ACAAGTTTGTACAAAAAAGCTGAACGAG- AAACGTAAANTGNTATAAATATCAATATATTAAATTAGATTTTGCAT- AAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCA- CTATG, or a nucleotide sequence complementary to the nucleotide sequence set forth in Figure 9 for αttRl, or mutants, fragments, variants or derivatives thereof As noted above for attBl, certain mutations, insertions, or deletions of one or more bases in the αttRl sequence contained in the nucleic acid molecules of the invention may be made without compromising the structural and functional integrity of these molecules, hence, nucleic acid molecules comprising such mutations, insertions, or deletions in the αttRl sequence are encompassed within the scope of the invention In another related aspect, the invention provides nucleic acid molecules comprising one or more nucleotide sequences encoding αttR2, or mutants, fragments, variants or derivatives thereof Such nucleic acid molecules may comprise an αttR2 nucleotide sequence having the sequence set forth in Figure 9, such as GCAGGTCGACCATAGTGACTGGATAT- GTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTA- ATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTT- TCTTGTACAAAGTGGT, or a nucleotide sequence complementary to the nucleotide sequence set forth in Figure 9 for αttR2, or mutants, fragments, variants or derivatives thereof As noted above for αttB 1 , certain mutations, insertions, or deletions of one or more bases in the αttR2 sequence contained in the nucleic acid molecules of the invention may be made without compromising the structural and functional integrity of these molecules, hence, nucleic acid molecules comprising such mutations, insertions, or deletions in the αttR2 sequence are encompassed within the scope of the invention Recombinant host cell strains containing attRl sites apposed to cloning sites in reading frame A, reading frame B, and reading frame C, E. coli DB3 l(pΕZC15101) (reading frame A, see Figure 64A), E. coli DB3 l(pEZC15102) (reading frame B, see Figure 64B), and E. coli DB3 l(pEZC15103) (reading frame C, see Figure 64C), and containing corresponding attR2 sites, were deposited on February 27, 1999, with the Collection, Agricultural Research Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA, as Deposit Nos NRRL B-30103, NRRL B-30104, and NRRL B-30105, respectively The attRl and attR2 sites within the deposited nucleic acid molecules are contained in nucleic acid cassettes in association with one or more additional functional sequences as described in more detail below In another related aspect, the invention provides nucleic acid molecules comprising one or more nucleotide sequences encoding ttLl, or mutants, fragments, variants and derivatives thereof Such nucleic acid molecules may comprise an αttLl nucleotide sequence having the sequence set forth in Figure 9, such as CAA ATA ATG ATT TTA TTT TGA CTG ATA GTG ACC TGT TCG TTG CAA CAA ATT GAT AAG CAA TGC TTT TTT ATA ATG CCA ACT TTG TAC AAA AAA GCA GGC T, or a nucleotide sequence complementary to the nucleotide sequence set forth in Figure 9 for αttL 1 , or mutants, fragments, variants or derivatives thereof As noted above for αttBl, certain mutations, insertions, or deletions of one or more bases in the αttLl sequence contained in the nucleic acid molecules of the invention may be made without compromising the structural and functional integrity of these molecules, hence, nucleic acid molecules comprising such mutations, insertions, or deletions in the αttLl sequence are encompassed within the scope of the invention In another related aspect, the invention provides nucleic acid molecules comprising one or more nucleotide sequences encoding αttL2, or mutants, fragments, variants and derivatives thereof Such nucleic acid molecules may comprise an αttL2 nucleotide sequence having the sequence set forth in Figure 9, such as C AAA TAA TGA TTT TAT TTT GAC TGA TAG TGA CCT GTT CGT TGC AAC AAA TTG ATA AGC AAT GCT TTC TTA TAA TGC CAA CTT TGT ACA AGA AAG CTG GGT, or a nucleotide sequence complementary to the nucleotide sequence set forth in Figure 9 for αttL2, or mutants, fragments, variants or derivatives thereof As noted above for αttBl, certain mutations, insertions, or deletions of one or more bases in the αttL2 sequence contained in the nucleic acid molecules of the invention may be made without compromising the structural and functional integrity of these molecules, hence, nucleic acid molecules comprising such mutations, insertions, or deletions in the αttL2 sequence are encompassed within the scope of the invention Recombinant host cell strains containing attLl sites apposed to cloning sites in reading frame A, reading frame B, and reading frame C, E. coli DB3 1 (pENTRIA) (reading frame A, see Figure 10), E. coli DB3 l(pENTR2B) (reading frame B, see Figure 11), andE. colιDB3 l(pΕNTR3C) (reading frame C, see Figure 12), and containing corresponding attL2 sites, were deposited on February 27, 1999, with the Collection, Agricultural Research Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA, as Deposit Nos NRRL B-30100, NRRL B-30101, and NRRL B-30102, respectively The attLl and attL2 sites within the deposited nucleic acid molecules are contained in nucleic acid cassettes in association with one or more additional functional sequences as described in more detail below Each of the recombination site sequences described herein or portions thereof, or the nucleotide sequence cassettes contained in the deposited clones, may be cloned or inserted into a vector of interest (for example, using the recombinational cloning methods described herein and/or standard restriction cloning techniques that are routine in the art) to generate, for example, Entry Vectors or Destination Vectors which may be used to transfer a desired segment of a nucleic acid molecule of interest (e.g. , a gene, cDNA molecule, or cDNA library) into a desired vector or into a host cell Using the information provided herein, such as the nucleotide sequences for the recombination site sequences described herein, an isolated nucleic acid molecule of the present invention encoding one or more recombination sites or portions thereof may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material Preferred such methods include PCR-based cloning methods, such as reverse transcriptase-PCR (RT-PCR) using primers such as those described herein and in the Examples below. Alternatively, vectors comprising the cassettes containing the recombination site sequences described herein are available commercially from Life Technologies, Inc. (Rockville, MD). The invention is also directed to nucleic acid molecules comprising one or more of the recombination site sequences or portions thereof and one or more additional nucleotide sequences, which may encode functional or structural sites such as one or more multiple cloning sites, one or more transcription termination sites, one or more transcriptional regulatory sequences (which may be promoters, enhancers, repressors, and the like), one or more translational signals (e.g., secretion signal sequences), one or more origins of replication, one or more fusion partner peptides (particularly glutathione S-transferase (GST), hexahistidine (His 6 ), and thioredoxin (Trx)), one or more selection markers or modules, one or more nucleotide sequences encoding localization signals such as nuclear localization signals or secretion signals, one or more origins of replication, one or more protease cleavage sites, one or more genes or portions of genes encoding a protein or polypeptide of interest, and one or more 5' polynucleotide extensions (particularly an extension of guanine residues ranging in length from about 1 to about 20, from about 2 to about 15, from about 3 to about 10, from about 4 to about 10, and most preferably an extension of 4 or 5 guanine residues at the 5' end of the recombination site nucleotide sequence. The one or more additional functional or structural sequences may or may not flank one or more of the recombination site sequences contained on the nucleic acid molecules of the invention. In some nucleic acid molecules of the invention, the one or more nucleotide sequences encoding one or more additional functional or structural sites may be operably linked to the nucleotide sequence encoding the recombination site. For example, certain nucleic acid molecules of the invention may have a promoter sequence operably linked to a nucleotide sequence encoding a recombination site or portion thereof of the invention, such as a T7 promoter, a phage lambda PL promoter, an E. coli lac, trp or tac promoter, and other suitable promoters which will be familiar to the skilled artisan. Nucleic acid molecules of the present invention, which may be isolated nucleic acid molecules, may be in the form of RNN such as mRΝN or in the form of DΝN including, for instance, cDΝA and genomic DΝA obtained by cloning or produced synthetically, or in the form of DΝA-RΝA hybrids. The nucleic acid molecules of the invention may be double-stranded or single-stranded. Single-stranded DΝA or RΝA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. The nucleic acid molecules of the invention may also have a number of topologies, including linear, circular, coiled, or supercoiled. By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule, DΝA or RΝA, which has been removed from its native environment. For example, recombinant DΝA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DΝA molecules include recombinant DΝA molecules maintained in heterologous host cells, and those DΝA molecules purified (partially or substantially) from a solution whether produced by recombinant DΝA or synthetic chemistry techniques. Isolated RΝA molecules include in vivo or in vitro RΝA transcripts of the DΝA molecules of the present invention. The present invention further relates to mutants, fragments, variants and derivatives of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of one or more recombination sites. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (see Lewin, B., ed., Genes II, , John Wiley & Sons, New York (1985)). Non-naturally occurring variants may be produced using art-known mutagenesis techniques, such as those described hereinbelow. Such variants include those produced by nucleotide substitutions, deletions or additions or portions thereof, or combinations thereof. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the encoded polypeρtide(s) or portions thereof, and which also do not substantially alter the reactivities of the recombination site nucleic acid sequences in recombination reactions. Also especially preferred in this regard are conservative substitutions. Particularly preferred mutants, fragments, variants, and derivatives of the nucleic acid molecules of the invention include, but are not limited to, insertions, deletions or substitutions of one or more nucleotide bases within the 15 bp core region (GCTTTTTTATACTAA) which is identical in all four wildtype lambda att sites, αttB, ttP, αttL and ttR (see U.S. Application Nos. 08/663,002, filed June 7, 1996 (now U.S. Patent No. 5,888,732), 09/005,476, filed January 12, 1998, and 09/177,387, filed October 23, 1998, which describes the core region in further detail, and the disclosures of which are incorporated herein by reference in their entireties). Analogously, the core regions in αttB 1 , αttP 1 , αttL 1 and αttRl are identical to one another, as are the core regions in αttB2, αttP2, αttL2 and αttR2. Particularly preferred in this regard are nucleic acid molecules comprising insertions, deletions or substitutions of one or more nucleotides within the seven bp overlap region (TTT AT AC, which is defined by the cut sites for the integrase protein and is the region where strand exchange takes place) that occurs within this 15 bp core region (GCTTTTTTATACTAA). Examples of such preferred mutants, fragments, variants and derivatives according to this aspect of the invention include, but are not limited to, nucleic acid molecules in which the thymine at position 1 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; in which the thymine at position 2 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; in which the thymine at position 3 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; in which the adenine at position 4 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or thymine; in which the thymine at position 5 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; in which the adenine at position 6 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or thymine; and in which the cytosine at position 7 of the seven bp overlap region has been deleted or substituted with a guanine, thymine, or adenine; or any combination of one or more such deletions and/or substitutions within this seven bp overlap region. As described in detail in Example 21 herein, mutants of the nucleic acid molecules of the invention in which substitutions have been made within the first three positions of the seven bp overlap (TTT AT AC) have been found in the present invention to strongly affect the specificity of recombination, mutant nucleic acid molecules in which substitutions have been made in the last four positions (TTTATAC) only partially alter recombination specificity, and mutant nucleic acid molecules comprising nucleotide substitutions outside of the seven bp overlap, but elsewhere within the 15 bp core region, do not affect specificity of recombination but do influence the efficiency of recombination. Hence, in an additional aspect, the present invention is also directed to nucleic acid molecules comprising one or more recombination site nucleotide sequences that affect recombination specificity, particularly one or more nucleotide sequences that may correspond substantially to the seven base pair overlap within the 15 bp core region, having one or more mutations that affect recombination specificity. Particularly preferred such molecules may comprise a consensus sequence (described in detail in Example 21 herein) such as NNNATAC, wherein "N" refers to any nucleotide (i.e., may be A, G, T U or C), with the proviso that if one of the first three nucleotides in the consensus sequence is a T/U, then at least one of the other two of the first three nucelotides is not a T/U. In a related aspect, the present invention is also directed to nucleic acid molecules comprising one or more recombination site nucleotide sequences that enhance recombination efficiency, particularly one or more nucleotide sequences that may correspond substantially to the core region and having one or more mutations that enhance recombination efficiency. By sequences or mutations that "enhance recombination efficiency" is meant a sequence or mutation in a recombination site, preferably in the core region (e.g., the 15 bp core region of αtt recombination sites), that results in an increase in cloning efficiency (typically measured by determining successful cloning of a test sequence, e.g., by determining CFU/ml for a given cloning mixture) when recombining molecules comprising the mutated sequence or core region as compared to molecules that do not comprise the mutated sequence or core region (e.g., those comprising a wildtype recombination site core region sequence) More specifically, whether or not a given sequence or mutation enhances recombination efficiency may be determined using the sequence or mutation in recombinational cloning as described herein, and determining whether the sequence or mutation provides enhanced recombinational cloning efficiency when compared to a non-mutated (e-g-, wildtype) sequence Methods of determining preferred cloning efficiency- enhancing mutations for a number of recombination sites, particularly for att recombination sites, are described herein, for example in Examples 22-25 Examples of preferred such mutant recombination sites include but are not limited to the αttL consensus core sequence of caacttnntnnnannaagttg (wherein "n" represents any nucleotide), for example the αttL5 sequence agcctgctttattatactaagttggcatta and the attL6 sequence ag c ct g ct tt ttt at attaagttggc att a , th e a ttB l 6 s e q u e n c e ggggacaactttgtacaaaaaagttggct , the αttB 2 2 sequence ggggacaactttgtacaagaaagctgggt, and the αttB2 10 sequence ggggacaactttgtacaagaaagttgggt Those of skill in the art will appreciate that, in addition to the core region, other portions of the att site may affect the efficiency of recombination There are five so-called arm binding sites for the integrase protein in the bacteriophage lambda attP site, two in attR (PI and P2), and three in attL (P' l, P'2 and P'3) Compared to the core binding sites, the integrase protein binds to arm sites with high affinity and interacts with core and arm sites through two different domains of the protein As with the core binding site a consensus sequence for the arm binding site consisting of C/AAGTCACTAT has been inferred from sequence comparison of the five arm binding sites and seven non-att sites (Ross and Landy, Proc. Natl. Acad. Sci. USA 79 7724-7728 ( 1982)) Each arm site has been mutated and tested for its effect in the excision and integration reactions (Numrych etal, Nucl. Acids Res. 18 3953 (1990)) Hence, specific sites are utilized in each reaction in different ways, namely, the PI and P'3 sites are essential for the integration reaction whereas the other three sites are dispensable to the integration reaction to varying degrees. Similarly, the P2, P' l and P'2 sites are most important for the excision reaction, whereas PI and P'3 are completely dispensable. Interestingly, when P2 is mutated the integration reaction occurs more efficiently than with the wild type attP site. Similarly, when PI and P'3 are mutated the excision reaction occurs more efficiently. The stimulatory effect of mutating integrase arm binding sites can be explained by removing sites that compete or inhibit a specific recombination pathway or that function in a reaction that converts products back to starting substrates. In fact there is evidence for an XlS-independent LR reaction (Abremski and Gottesman, J. Mol. Biol. 153:61-1% (1981)). Thus, in addition to modifications in the core region of the att site, the present invention contemplates the use of att sites containing one or more modifications in the integrase arm-type binding sites. In some preferred embodiments, one or more mutations may be introduced into one or more of the P 1 , P' 1 , P2, P'2 and P'3 sites. In some preferred embodiments, multiple mutations may be introduced into one or more of these sites. Preferred such mutations include those which increase the recombination in vitro. For example, in some embodiments mutations may be introduced into the arm-type binding sites such that integrative recombination, corresponding to the BP reaction, is enhanced. In other embodiments, mutations may be introduced into the arm-type binding sites such that excisive recombination, corresponding to the LR reaction, is enhanced. Of course, based on the guidance contained herein, particularly in the construction and evaluation of effects of mutated recombination sites upon recombinational specificity and efficiency, analogous mutated or engineered sequences may be produced for other recombination sites described herein (including but not limited to lox, FRT, and the like) and used in accordance with the invention. For example, much like the mutagenesis strategy used to select core binding sites that enhance recombination efficiency, similar strategies can be employed to select changes in the arms of attP, attL and attR, and in analogous sequences in other recombination sites such as lox, FRT and the like, that enhance recombination efficiency. Hence, the construction and evaluation of such mutants is well within the abilities of those of ordinary skill in the art without undue experimentation. One suitable methodology for preparing and evaluating such mutations is found in Numrych, et al , (1990) Nucleic Acids Research 18(13) 3953-3959 Other mutant sequences and nucleic acid molecules that may be suitable to enhance recombination efficiency will be apparent from the description herein, or may be easily determined by one of ordinary skill using only routine experimentation in molecular biology in view of the description herein and information that is readily available in the art Since the genetic code is well known in the art, it is also routine for one of ordinary skill in the art to produce degenerate variants of the nucleic acid molecules described herein without undue experimentation Hence, nucleic acid molecules comprising degenerate variants of nucleic acid sequences encoding the recombination sites described herein are also encompassed within the scope of the invention Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequences of the seven bp overlap region within the 15 bp core region of the recombination sites described herein, or the nucleotide sequences of αttB 1 , αttB2, αttP 1 , αttP2, αttL 1 , αttL2, αttRl or αttR2 as set forth in Figure 9 (or portions thereof), or a nucleotide sequence complementary to any of these nucleotide sequences, or fragments, variants, mutants, and derivatives thereof By a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a particular recombination site or portion thereof is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations (e.g. , insertions, substitutions, or deletions) per each 100 nucleotides of the reference nucleotide sequence encoding the recombination site For example, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference αttBl nucleotide sequence, up to 5% of the nucleotides in the αttBl reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the αttBl reference sequence may be inserted into the αttB 1 reference sequence These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence As a practical matter, whether any particular nucleic acid molecule is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, a given recombination site nucleotide sequence or portion thereof can be determined conventionally using known computer programs such as DNAsis software (Hitachi Software, San Bruno, California) for initial sequence alignment followed by ESEE version 3 0 DN A/protein sequence software (cabot@trog mbb sfu ca) for multiple sequence alignments Alternatively, such determinations may be accomplished using the BESTFIT program (Wisconsin Sequence Analysis Package, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711), which employs a local homology algorithm (Smith and Waterman, Advances in Applied Mathematics 2 482-489 (1981)) to find the best segment of homology between two sequences When using DNAsis, ESEE, BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed The present invention is directed to nucleic acid molecules at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the αttBl, αttB2, αttP 1 , αttP2, αttL 1 , αttL2, αttRl or αttR2 nucleotide sequences as set forth in Figure 9, or to the nucleotide sequence of the deposited clones, irrespective of whether they encode particular functional polypeptides This is because even where a particular nucleic acid molecule does not encode a particular functional polypeptide, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer Mutations can also be introduced into the recombination site nucleotide sequences for enhancing site specific recombination or altering the specificities of the reactants, etc Such mutations include, but are not limited to recombination sites without translation stop codons that allow fusion proteins to be encoded, recombination sites recognized by the same proteins but differing in base sequence such that they react largely or exclusively with their homologous partners allowing multiple reactions to be contemplated, and mutations that prevent hairpin formation of recombination sites Which particular reactions take place can be specified by which particular partners are present in the reaction mixture There are well known procedures for introducing specific mutations into nucleic acid sequences A number of these are described in Ausubel, F M et al, Current Protocols in Molecular Biology, Wiley Interscience, New York (1989- 1996) Mutations can be designed into oligonucleotides, which can be used to modify existing cloned sequences, or in amplification reactions Random mutagenesis can also be employed if appropriate selection methods are available to isolate the desired mutant DNA or RNA The presence of the desired mutations can be confirmed by sequencing the nucleic acid by well known methods The following non-limiting methods can be used to modify or mutate a given nucleic acid molecule encoding a particular recombination site to provide mutated sites that can be used in the present invention 1 By recombination of two parental DNA sequences by site-specific (e g attL and attR to give attP) or other (e g homologous) recombination mechanisms where the parental DNA segments contain one or more base alterations resulting in the final mutated nucleic acid molecule, 2 By mutation or mutagenesis (site-specific, PCR, random, spontaneous, etc) directly of the desired nucleic acid molecule, 3 By mutagenesis (site-specific, PCR, random, spontaneous, etc) of parental DNA sequences, which are recombined to generate a desired nucleic acid molecule, 4 By reverse transcription of an RNA encoding the desired core sequence, and 5 By de novo synthesis (chemical synthesis) of a sequence having the desired base changes, or random base changes followed by sequencing or functional analysis according to methods that are routine in the art The functionality of the mutant recombination sites can be demonstrated in ways that depend on the particular characteristic that is desired For example, the lack of translation stop codons in a recombination site can be demonstrated by expressing the appropriate fusion proteins Specificity of recombination between homologous partners can be demonstrated by introducing the appropriate molecules into in vitro reactions, and assaying for recombination products as described herein or known in the art Other desired mutations in recombination sites might include the presence or absence of restriction sites, translation or transcription start signals, protein binding sites, particular coding sequences, and other known functionalities of nucleic acid base sequences Genetic selection schemes for particular functional attributes in the recombination sites can be used according to known method steps For example, the modification of sites to provide (from a pair of sites that do not interact) partners that do interact could be achieved by requiring deletion, via recombination between the sites, of a DNA sequence encoding a toxic substance Similarly, selection for sites that remove translation stop sequences, the presence or absence of protein binding sites, etc , can be easily devised by those skilled in the art Accordingly, the present invention also provides a nucleic acid molecule, comprising at least one DNA segment having at least one, and preferably at least two, engineered recombination site nucleotide sequences of the invention flanking a selectable marker and/or a desired DNA segment, wherein at least one of said recombination site nucleotide sequences has at least one engineered mutation that enhances recombination in vitro in the formation of a Cointegrate DNA or a Product DNA Such engineered mutations may be in the core sequence of the recombination site nucleotide sequence of the invention, see U S Application Nos 08/486,139, filed June 7, 1995, 08/663,002, filed June 7, 1996 (now U S Patent No 5,888,732), 09/005,476, filed January 12, 1998, and 09/177,387, filed October 23, 1998, the disclosures of which are all incorporated herein by reference in their entireties While in the preferred embodiment the recombination sites differ in sequence and do not interact with each other, it is recognized that sites comprising the same sequence, which may interact with each other, can be manipulated or engineered to inhibit recombination with each other Such conceptions are considered and incorporated herein For example, a protein binding site (e.g. , an antibody-binding site, a histone-binding site, an enzyme-binding site, or a binding site for any nucleic acid molecule-binding protein) can be engineered adjacent to one of the sites In the presence of the protein that recognizes the engineered site, the recombinase fails to access the site and another recombination site in the nucleic acid molecule is therefore used preferentially In the cointegrate this site can no longer react since it has been changed, e.g., from attB to attL During or upon resolution of the cointegrate, the protein can be inactivated (e.g., by antibody, heat or a change of buffer) and the second site can undergo recombination The nucleic acid molecules of the invention can have at least one mutation that confers at least one enhancement of said recombination, said enhancement selected from the group consisting of substantially (i) favoring integration, (ii) favoring recombination, (ii) relieving the requirement for host factors, (iii) increasing the efficiency of said Cointegrate DNA or Product DNA formation, (iv) increasing the specificity of said Cointegrate DNA or Product DNA formation, and (v) adding or deleting protein binding sites In other embodiments, the nucleic acid molecules of the invention may be PCR primer molecules, which comprise one or more of the recombination site sequences described herein or portions thereof, particularly those shown in Figure 9 (or sequences complementary to those shown in Figure 9), or mutants, fragments, variants or derivatives thereof, attached at the 3' end to a target- specific template sequence which specifically interacts with a target nucleic acid molecule which is to be amplified Primer molecules according to this aspect of the invention may further comprise one or more, (e.g., 1, 2, 3, 4, 5, 10, 20, 25, 50, 100, 500, 1000, or more) additional bases at their 5' ends, and preferably comprise one or more (particularly four or five) additional bases, which are preferably guanines, at their 5' ends, to increase the efficiency of the amplification products incorporating the primer molecules in the recombinational cloning system of the invention Such nucleic acid molecules and primers are described in detail in the examples herein, particularly in Examples 22-25 Certain primers of the invention may comprise one or more nucleotide deletions in the αttB 1 , αttB2, αttP 1 , αttP2, αttL 1 , αttL2, αttRl or αttR2 sequences as set forth in Figure 9 In one such aspect, for example, αttB2 primers may be constructed in which one or more of the first four nucleotides at the 5' end of the αttB2 sequence shown in Figure 9 have been deleted Primers according to this aspect of the invention may therefore have the sequence (αttB2(-l)) CCCAGCTTTCTTGTACAAAGTGGTnnnnnnnnnnnnn n (αttB2(-2)) CCAGCTTTCTTGTACAAAGTGGTnnnnnnnnnnnnnn n (αttB2(-3)) CAGCTTTCTTGTACAAAGTGGTnnnnnnnnnnnnnnn n (αttB2(-4)) AGCTTTCTTGTACAANGTGGTnnnnnnnnnnnnnnnn n, wherein "nnnnnnnnnnnnn n" at the 3' end of the primer represents a target- specific sequence of any length, for example from one base up to all of the bases of a target nucleic acid molecule (e.g., a gene) or a portion thereof, the sequence and length which will depend upon the identity of the target nucleic acid molecule which is to be amplified The primer nucleic acid molecules according to this aspect of the invention may be produced synthetically by attaching the recombination site sequences depicted in Figure 9, or portions thereof, to the 5' end of a standard PCR target- specific primer according to methods that are well-known in the art Alternatively, additional primer nucleic acid molecules of the invention may be produced synthetically by adding one or more nucleotide bases, which preferably correspond to one or more, preferably five or more, and more preferably six or more, contiguous nucleotides of the att nucleotide sequences described herein (see, e.g. , Example 20 herein, see also U S Application Νos 08/663,002, filed June 7, 1996 (now U S Patent No 5,888,732), 09/005,476, filed January 12, 1998, and 09/177,387, filed October 23, 1998, the disclosures of which are all incorporated herein by reference in their entireties), to the 5' end of a standard PCR target- specific primer according to methods that are well-known in the art, to provide primers having the specific nucleotide sequences described herein As noted above, primer nucleic acid molecules according to this aspect of the invention may also optionally comprise one, two, three, four, five, or more additional nucleotide bases at their 5' ends, and preferably will comprise four or five guanines at their 5' ends In one particularly preferred such aspect, the primer nucleic acid molecules of the invention may comprise one or more, preferably five or more, more preferably six or more, still more preferably 6-18 or 6-25, and most preferably 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, contiguous nucleotides or bp of the αttBl or αttB2 nucleotide sequences depicted in Figure 9 (or nucleotides complementary thereto), linked to the 5' end of a target-specific (e.g. , a gene-specific) primer molecule Primer nucleic acid molecules according to this aspect of the invention include, but are not limited to, αttBl- and αttB2-derived primer nucleic acid molecules having the following nucleotide sequences ACAAGTTTGTACAAAAAAGCAGGCT-nnnnnnnnnnnnn n ACCACTTTGTACAAGAANGCTGGGT-nnnnnnnnnnnnn n TGTACAAAAAAGCAGGCT-nnnnnnnnnnnnn n TGTACAAGANAGCTGGGT-nnnnnnnnnnnnn n ACAANAAAGCAGGCT-nnnnnnnnnnnnn n ACAAGAAAGCTGGGT-nnnnnnnnnnnnn n AANAAGCAGGCT-nnnnnnnnnnnnn n AGAANGCTGGGT-nnnnnnnnnnnnn n ANAAGCAGGCT-nnnnnnnnnnnnn n GAANGCTGGGT-nnnnnnnnnnnnn n AAAGCAGGCT-nnnnnnnnnnnnn n AAAGCTGGGT-nnnnnnnnnnnnn n AAGCAGGCT-nnnnnnnnnnnnn n AAGCTGGGT-nnnnnnnnnnnnn n AGCAGGCT-nnnnnnnnnnnnn n AGCTGGGT-nnnnnnnnnnnnn n GCAGGCT-nnnnnnnnnnnnn n GCTGGGT-nnnnnnnnnnnnn n CAGGCT-nnnnnnnnnnnnn n CTGGGT-nnnnnnnnnnnnn n, wherein "nnnnnnnnnnnnn n" at the 3' end of the primer represents a target- specific sequence of any length, for example from one base up to all of the bases of a target nucleic acid molecule (e.g., a gene) or a portion thereof, the sequence and length which will depend upon the identity of the target nucleic acid molecule which is to be amplified Of course, it will be apparent to one of ordinary skill from the teachings contained herein that additional primer nucleic acid molecules analogous to those specifically described herein may be produced using one or more, preferably five or more, more preferably six or more, still more preferably ten or more, 15 or more, 20 or more, 25 or more, 30 or more, etc (through to and including all) of the contiguous nucleotides or bp of the αttP 1 , αttP2, αttL 1 , αttL2, αttRl or αttR2 nucleotide sequences depicted in Figure 9 (or nucleotides complementary thereto), linked to the 5' end of a target-specific (e.g. , a gene-specific) primer molecule As noted above, such primer nucleic acid molecules may optionally further comprise one, two, three, four, five, or more additional nucleotide bases at their 5' ends, and preferably will comprise four guanines at their 5' ends Other primer molecules comprising the αttBl, αttB2, αttPl, αttP2, αttLl, αttL2, αttRl and αttR2 sequences depicted in Figure 9, or portions thereof, may be made by one of ordinary skill without resorting to undue experimentation in accordance with the guidance provided herein The primers of the invention described herein are useful in producing PCR fragments having a nucleic acid molecule of interest flanked at each end by a recombination site sequence (as described in detail below in Example 9), for use in cloning of PCR-amplified DNA fragments using the recombination system of the invention (as described in detail below in Examples 8, 19 and 21-25) Vectors The invention also relates to vectors comprising one or more of the nucleic acid molecules of the invention, as described herein In accordance with the invention, any vector may be used to construct the vectors of the invention In particular, vectors known in the art and those commercially available (and variants or derivatives thereof) may in accordance with the invention be engineered to include one or more nucleic acid molecules encoding one or more recombination sites (or portions thereof), or mutants, fragments, or derivatives thereof, for use in the methods of the invention Such vectors may be obtained from, for example, Vector Laboratories Inc , InVitrogen, Promega, Novagen, New England Biolabs, Clontech, Roche, Pharmacia, EpiCenter, OriGenes Technologies Inc , Stratagene, Perkin Elmer, Pharmingen, Life Technologies, Inc , and Research Genetics Such vectors may then for example be used for cloning or subcloning nucleic acid molecules of interest General classes of vectors of particular interest include prokaryotic and/or eukaryotic cloning vectors, Expression Vectors, fusion vectors, two-hybrid or reverse two-hybrid vectors, shuttle vectors for use in different hosts, mutagenesis vectors, transcription vectors, vectors for receiving large inserts and the like Other vectors of interest include viral origin vectors (Ml 3 vectors, bacterial phage A vectors, bacteriophage PI vectors, adenovirus vectors, herpesvirus vectors, retrovirus vectors, phage display vectors, combinatorial library vectors), high, low, and adjustable copy number vectors, vectors which have compatible replicons for use in combination in a single host (pACYC184 and pBR322) and eukaryotic episomal replication vectors (pCDM8) Particular vectors of interest include prokaryotic Expression Vectors such as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisN B, and C, pRSET A, B, and C (Invitrogen, Inc ), pGEMEX-1, and pGEMEX-2 (Promega, Inc ), the pET vectors (Νovagen, Inc ), pTrc99N pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia, Inc ), pKK233-2 and pKK388-l (Clontech, Inc ), and pProEx-HT (Life Technologies, Inc ) and variants and derivatives thereof Destination Vectors can also be made from eukaryotic Expression Vectors such as pFastBac, pFastBac HT, pFastBac DUAL, pSFV, and pTet- Splice (Life Technologies, Inc ), pEUK-Cl, pPUR, pMAM, pMAMneo, pBHOl, pBI121, pDR2, pCMVEBΝN and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCHl 10, and pKK232-8 (Pharmacia, Inc ), p3'SS, pXTl, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc ), and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBsueBacIII, pCDM8, pcDNAl, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Inc.) and variants or derivatives thereof Other vectors of particular interest include pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), MACs (mammalian artificial chromosomes), pQE70, pQE60, pQE9 (Quiagen), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8N pΝH16N pΝH18N pΝH46A (Stratagene), pcDNA3 (InVitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT 1, pSPORT2, pCMVSPORT2.0andpSV- SPORT1 (Life Technologies, Inc.) and variants or derivatives thereof. Additional vectors of interest include pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1 (-)/Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZα, pGAPZ, pGAPZα, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SPl), pVgRXR, pcDNA2.1. pYES2, pZErOl . l, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNAl .l, pcDNAl .1/Amp, pcDNA3.1 , pcDNA3.1/Zeo, pSe, S V2, pRc/CMV2, pRc/RSV, pREP4, pREP7, pREP8, pREP9, pREPIO, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen; λExCell, λgtl 1, pTrc99A, pKK223-3, pGEX-lλT, pGEX-2T, pGEX-2TK, pGEX-4T-l, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-l, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCHl lO, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia; pSCREEN-lb(+), pT7Blue(R), pT7Blue-2, pCITE- 4abc(+), pOCUS-2, pTAg, pET-32 LIC, pET-30 LIC, pBAC-2cp LIC, pBACgus- 2cp LIC, pT7Blue-2 LIC, pT7Blue-2, λSCREEN-1, ABlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pETl labcd, pET12abc, pET-14b, pET-15b, pET-16b, pET- 17b- pET- 17xb, pET- 19b, pET-20b(+), pET-21 abcd(+), pET-22b(+), pET- 23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET- 28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET- 33b(+), pBAC-1, pBACgus-1, pBAC4x-l, pBACgus4x-l, pBAC-3cp, pBACgus- 2cp, pBACsurf-1, pig, Signal pig, pYX, Selecta Vecta-Neo, Selecta Vecta - Hyg, and Selecta Vecta - Gpt from Novagen; pLexN pB42AD, pGBT9, pAS2-l, pGAD424, pACT2, pGAD GL, pGAD GH, pGADIO, pGilda, pEZM3, pEGFP, pEGFP- 1 , pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2- Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic, pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMVβ, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRESlneo, pIRESlhyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX 4T- 1/2/3, pYEX-S 1 , pBacPAK-His, pBacPAK8/9, pAcUW31 , BacPAKό, pTriplEx, λgtlO, λgtl 1, pWE15, and λTriplEx from Clontech, Lambda ZAP II, pBK-CMV, pBK-RS V, pBluescript II KS +/-, pBluescript II SK +/-, p AD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS +/-, pBC KS +/-, pBC SK +/-, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-l labcd, pSPUTK, pESP-1, pCMVLad, pOPRSVI/MCS, pOPI3 CAT, pXTl, pSG5, pPbac, pMbac, pMClneo, pMClneo Poly A, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 from Stratagene Two-hybrid and reverse two-hybrid vectors of particular interest include pPC86, pDBLeu, pDBTrp, pPC97, p2 5, pGADl-3, pGADIO, pACt, pACT2, pGADGL, pGADGH, pAS2-l, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pfflSi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants or derivatives thereof Yeast Expression Vectors of particular interest include pESP-1, pESP-2, pESC-His, pESC-Trp, pESC-URA, pESC-Leu (Stratagene), pRS401, pRS402, pRS41 1, pRS412, pRS421, pRS422, and variants or derivatives thereof According to the invention, the vectors comprising one or more nucleic acid molecules encoding one or more recombination sites, or mutants, variants, fragments, or derivatives thereof, may be produced by one of ordinary skill in the art without resorting to undue experimentation using standard molecular biology methods For example, the vectors of the invention may be produced by introducing one or more of the nucleic acid molecules encoding one or more recombination sites (or mutants, fragments, variants or derivatives thereof) into one or more of the vectors described herein, according to the methods described, for example, in Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). In a related aspect of the invention, the vectors may be engineered to contain, in addition to one or more nucleic acid molecules encoding one or more recombination sites (or portions thereof), one or more additional physical or functional nucleotide sequences, such as those encoding one or more multiple cloning sites, one or more transcription termination sites, one or more transcriptional regulatory sequences (e.g., one or more promoters, enhancers, or repressors), one or more selection markers or modules, one or more genes or portions of genes encoding a protein or polypeptide of interest, one or more translational signal sequences, one or more nucleotide sequences encoding a fusion partner protein or peptide (e.g. , GST, His 6 or thioredoxin), one or more origins of replication, and one or more 5' or 3' polynucleotide tails (particularly a poly-G tail). According to this aspect of the invention, the one or more recombination site nucleotide sequences (or portions thereof) may optionally be operably linked to the one or more additional physical or functional nucleotide sequences described herein. Preferred vectors according to this aspect of the invention include, but are not limited to: pENTRIA (Figures lOAand 10B), pENTR2B (Figures 1 lAand 1 IB), pENTR3C (Figures 12A and 12B), pENTR4 (Figures 13A and 13B), pENTR5 (Figures 14A and 14B), pENTRό (Figures 15Aand 15B), pENTR7 (Figures 16A and 16B), pENTR8 (Figures 17A and 17B), pENTR9 (Figures 18A and 18B), pENTRlO (Figures 19A and 19B), pENTRl l (Figures 20A and 20B), pDESTl (Figures 21A-D), pDEST2 (Figure 22A-D), pDEST3 (Figure 23A-D), pDEST4 (Figure 24A-D), pDEST5 (Figure 25A-D), pDEST6 (Figure 26A-D), pDEST7 (Figure 27A-C), pDEST8 (Figure 28 A-D), pDEST9 (Figure 29A-E), pDEST 10 (Figure 30A-D), pDESTl l (Figure 31 A-D), pDEST12.2 (also known as pDEST12) (Figure 32A-D), pDEST13 (Figure 33A-C), pDEST14 (Figure 34A- D), pDEST15 (Figure 35A-D), pDESTlό (Figure 36A-D), pDEST17 (Figure 37 A-D), pDESTlδ (Figure 38 A-D), pDEST19 (Figure 39A-D), pDEST20 (Figure 40 A-D), pDEST21 (Figure 41 A-E), pDEST22 (Figure 42A-D), pDEST23 (Figure 43A-D), pDEST24 (Figure 44A-D), pDEST25 (Figure 45A-D), pDEST26 (Figure 46A-D), pDEST27 (Figure 47A-D), pEXP501 (also known as pCMVSPORTό) (Figure 48 A-B), pDONR201 (also known as pENTR21 attP vector or pAttPkan Donor Vector) (Figure 49), pDONR202 (Figure 50), pDONR203 (also known as pEZ15812) (Figure 51), pDONR204 (Figure 52), pDONR205 (Figure 53), pDONR206 (also known as pENTR22 attP vector or pAttPgen Donor Vector) (Figure 54), pMAB58 (Figure 87), pMAB62 (Figure 88), pDEST28 (Figure 90), pDEST29 (Figure 91 ), pDEST30 (Figure 92), pDEST31 (Figure 93), pDEST32 (Figure 94), pDEST33 (Figure 95), pDEST34 (Figure 96), pDONR207 (Figure 97), pMAB85 (Figure 98), pMAB86 (Figure 99), and fragments, mutants, variants, and derivatives thereof. However, it will be understood by one of ordinary skill that the present invention also encompasses other vectors not specifically designated herein, which comprise one or more of the isolated nucleic acid molecules of the invention encoding one or more recombination sites or portions thereof (or mutants, fragments, variants or derivatives thereof), and which may further comprise one or more additional physical or functional nucleotide sequences described herein which may optionally be operably linked to the one or more nucleic acid molecules encoding one or more recombination sites or portions thereof. Such additional vectors may be produced by one of ordinary skill according to the guidance provided in the present specification. Polymerases Preferred polypeptides having reverse transcriptase activity (i.e., those polypeptides able to catalyze the synthesis of a DNA molecule from an RNA template) for use in accordance with the present invention include, but are not limited to Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, Myeloblastosis Associated Virus (MAV) reverse transcriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase, retroviral reverse transcriptase, retrotransposon reverse transcriptase, hepatitis B reverse transcriptase, cauliflower mosaic virus reverse transcriptase and bacterial reverse transcriptase. Particularly preferred are those polypeptides having reverse transcriptase activity that are also substantially reduced in RNAse H activity (i.e., "RNAse H " " polypeptides) By a polypeptide that is "substantially reduced in RNase H activity" is meant that the polypeptide has less than about 20%, more preferably less than about 15%, 10% or 5%, and most preferably less than about 2%, of the RNase H activity of a wildtype or RNase H" enzyme such as wildtype M-MLV reverse transcriptase The RNase H activity may be determined by a variety of assays, such as those described, for example, in U S Patent No 5,244,797, in Kotewicz, M L et al, Nucl. Acids Res. 16 265 (1988) and in Gerard, G F , et al, FOCUS 14(5) 91 (1992), the disclosures of all of which are fully incorporated herein by reference Suitable RNAse H " polypeptides for use in the present invention include, but are not limited to, M-MLV H " reverse transcriptase, RSV H " reverse transcriptase, AMV H " reverse transcriptase, RAV H " reverse transcriptase, MAV H " reverse transcriptase, HIV H " reverse transcriptase, THERMOSCRIPT™ reverse transcriptase and THERMOSCRIPT™ II reverse transcriptase, and SUPERSCRIPT™ I reverse transcriptase and SUPERSCRIPT™ II reverse transcriptase, which are obtainable, for example, from Life Technologies, Inc (Rockville, Maryland) See generally published PCT application WO 98/47912 Other polypeptides having nucleic acid polymerase activity suitable for use in the present methods include thermophilic DNA polymerases such as DNA polymerase I, DNA polymerase III, Klenow fragment, T7 polymerase, and T5 polymerase, and thermostable DNA polymerases including, but not limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopohtana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus htorahs (Th or VENT®) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Pyrococcus species GB-D (or DEEPVENT®) DNA polymerase, Pyrococcus woosn (Pwo) DNA polymerase, Bacillus sterothermophilus (Bst) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME®) DNA polymerase, Methanobactenum thermoautotrophwum (Mth) DNA polymerase, and mutants, variants and derivatives thereof Such polypeptides are available commercially, for example from Life Technologies, Inc (Rockville, MD), New Englan BioLabs (Beverly, MA), and Sigma Aldrich (St Louis, MO) Host Cells The invention also relates to host cells comprising one or more of the nucleic acid molecules or vectors of the invention, particularly those nucleic acid molecules and vectors described in detail herein Representative host cells that may be used according to this aspect of the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells Preferred bacterial host cells include Escherichia spp cells (particularly E. coli cells and most particularly E. co strains DH10B, Stbl2, DH5α, DB3, DB3 1 (preferably E. coli LIBRARY EFFICIENCY® DB3 1™ Competent Cells, Life Technologies, Inc , Rockville, MD), DB4 and DB5, see U S Provisional Application No 60/122,392, filed on March 2, 1999, the disclosure of which is incorporated by reference herein in its entirety), Bacillus spp cells (particularly B. subtilis and B. megatenum cells), Streptomyces spp cells, Erwinia spp cells, Klebsiella spp cells, Serratia spp cells (particularly S. marcessans cells), Pseudomonas spp cells (particularly P. aeruginosa cells), and Salmonella spp cells (particularly S. typhimurium and S. typhi cells) Preferred animal host cells include insect cells (most particularly Drosophila melanogaster cells, Spodoptera frugiperda Sf9 and Sf21 cells and Trichoplusa High-Five cells), nematode cells (particularly C. elegans cells), avian cells, amphibian cells (particularly Xenopus laevis cells), reptilian cells, and mammalian cells (most particularly CHO, COS, VERO, BHK and human cells) Preferred yeast host cells include Saccharomyces cerevisiae cells and Pichia pastoris cells These and other suitable host cells are available commercially, for example from Life Technologies, Inc (Rockville, Maryland), American Type Culture Collection (Manassas, Virginia), and Agricultural Research Culture Collection (NRRL, Peoria, Illinois) Methods for introducing the nucleic acid molecules and/or vectors of the invention into the host cells described herein, to produce host cells comprising one or more of the nucleic acid molecules and/or vectors of the invention, will be familiar to those of ordinary skill in the art For instance, the nucleic acid molecules and/or vectors of the invention may be introduced into host cells using well known techniques of infection, transduction, transfection, and transformation The nucleic acid molecules and/or vectors of the invention may be introduced alone or in conjunction with other the nucleic acid molecules and/or vectors Alternatively, the nucleic acid molecules and/or vectors of the invention may be introduced into host cells as a precipitate, such as a calcium phosphate precipitate, or in a complex with a lipid Electroporation also may be used to introduce the nucleic acid molecules and/or vectors of the invention into a host Likewise, such molecules may be introduced into chemically competent cells such as E. coh If the vector is a virus, it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cells Hence, a wide variety of techniques suitable for introducing the nucleic acid molecules and/or vectors of the invention into cells in accordance with this aspect of the invention are well known and routine to those of skill in the art Such techniques are reviewed at length, for example, in Sambrook, J , et al, Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press, pp 16 30-16 55 (1989), Watson, J D , etal, Recombinant DNA, 2ndEd., New York W H Freeman and Co , pp 213-234 (1992), and Winnacker, E -L , From Genes to Clones, New York VCH Publishers (1987), which are illustrative of the many laboratory manuals that detail these techniques and which are incorporated by reference herein in their entireties for their relevant disclosures Polypeptides In another aspect, the invention relates to polypeptides encoded by the nucleic acid molecules of the invention (including polypeptides and amino acid sequences encoded by all possible reading frames of the nucleic acid molecules of the invention), and to methods of producing such polypeptides Polypeptides of the present invention include purified or isolated natural products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, insect, mammalian, avian and higher plant cells The polypeptides of the invention may be produced by synthetic organic chemistry, and are preferably produced by standard recombinant methods, employing one or more of the host cells of the invention comprising the vectors or isolated nucleic acid molecules of the invention According to the invention, polypeptides are produced by cultivating the host cells of the invention (which comprise one or more of the nucleic acid molecules of the invention, preferably contained within an Expression Vector) under conditions favoring the expression of the nucleotide sequence contained on the nucleic acid molecule of the invention, such that the polypeptide encoded by the nucleic acid molecule of the invention is produced by the host cell As used herein, "conditions favoring the expression of the nucleotide sequence" or "conditions favoring the production of a polypeptide" include optimal physical (e.g., temperature, humidity, etc ) and nutritional (e.g. , culture medium, ionic) conditions required for production of a recombinant polypeptide by a given host cell Such optimal conditions for a variety of host cells, including prokaryotic (bacterial), mammalian, insect, yeast, and plant cells will be familiar to one of ordinary skill in the art, and may be found, for example, in Sambrook, J , et al. , Molecular Cloning, A Laboratory Manual, 2nd Ed. , Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press, (1989), Watson, J D , et al , Recombinant DNA, 2nd Ed. , New York W H Freeman and Co , and Winnacker, E -L , From Genes to Clones, New York VCH Publishers (1987) In some aspects, it may be desirable to isolate or purify the polypeptides of the invention (e.g. , for production of antibodies as described below), resulting in the production of the polypeptides of the invention in isolated form The polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods of protein purification that are routine in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography For example, His6 or GST fusion tags on polypeptides made by the methods of the invention may be isolated using appropriate affinity chromatography matrices which bind polypeptides bearing His6 or GST tags, as will be familiar to one of ordinary skill in the art. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Isolated polypeptides of the invention include those comprising the amino acid sequences encoded by one or more of the reading frames of the polynucleotides comprising one or more of the recombination site-encoding nucleic acid molecules of the invention, including those encoding αttB 1 , αttB2, αttP 1 , αttP2, αttL 1 , αttL2, αttRl and αttR2 having the nucleotide sequences set forth in Figure 9 (or nucleotide sequences complementary thereto), or fragments, variants, mutants and derivatives thereof; the complete amino acid sequences encoded by the polynucleotides contained in the deposited clones described herein; the amino acid sequences encoded by polynucleotides which hybridize under stringent hybridization conditions to polynucleotides having the nucleotide sequences encoding the recombination site sequences of the invention as set forth in Figure 9 (or a nucleotide sequence complementary thereto); or a peptide or polypeptide comprising a portion or a fragment of the above polypeptides. The invention also relates to additional polypeptides having one or more additional amino acids linked (typically by peptidyl bonds to form a nascent polypeptide) to the polypeptides encoded by the recombination site nucleotide sequences or the deposited clones. Such additional amino acid residues may comprise one or more functional peptide sequences, for example one or more fusion partner peptides (e.g. , GST, His 6 , Trx, etc.) and the like. As used herein, the terms "protein," "peptide, ""oligopeptide" and "polypeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of two or more amino acids, preferably five or more amino acids, or more preferably ten or more amino acids, coupled by (a) peptidyl linkage(s), unless otherwise defined in the specific contexts below As is commonly recognized in the art, all polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus It will be recognized by those of ordinary skill in the art that some amino acid sequences of the polypeptides of the invention can be varied without significant effect on the structure or function of the polypeptides If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine structure and activity In general, it is possible to replace residues which form the tertiary structure, provided that residues performing a similar function are used In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the polypeptide Thus, the invention further includes variants of the polypeptides of the invention, including allelic variants, which show substantial structural homology to the polypeptides described herein, or which include specific regions of these polypeptides such as the portions discussed below Such mutants may include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule) Small changes or such "neutral" or "conservative" amino acid substitutions will generally have little effect on activity Typical conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He, interchange of the hydroxylated residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amidated residues Asn and Gin, exchange of the basic residues Lys and Arg, and replacements among the aromatic residues Phe and Tyr Thus, the fragment, derivative or analog of the polypeptides of the invention, such as those comprising peptides encoded by the recombination site nucleotide sequences described herein, may be (i) one in which one or more of the amino acid residues are substituted with a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), and such substituted amino acid residue may be encoded by the genetic code or may be an amino acid (e.g., desmosine, citrulline, ornithine, etc.) that is not encoded by the genetic code; (ii) one in which one or more of the amino acid residues includes a substituent group (e.g. , a phosphate, hydroxyl, sulfate or other group) in addition to the normal "R" group of the amino acid; (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which additional amino acids are fused to the mature polypeptide, such as an immunoglobulin Fc region peptide, a leader or secretory sequence, a sequence which is employed for purification of the mature polypeptide (such as GST) or a proprotein sequence. Such fragments, derivatives and analogs are intended to be encompassed by the present invention, and are within the scope of those skilled in the art from the teachings herein and the state of the art at the time of invention. The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. Recombinantly produced versions of the polypeptides of the invention can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). As used herein, the term "substantially purified" means a preparation of an individual polypeptide of the invention wherein at least 50%, preferably at least 60%, 70%, or 75% and more preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (by mass) of contaminating proteins (i.e., those that are not the individual polypeptides described herein or fragments, variants, mutants or derivatives thereof) have been removed from the preparation. The polypeptides of the present invention include those which are at least about 50% identical, at least 60% identical, at least 65% identical, more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical, to the polypeptides described herein. For example, preferred αttB l -containing polypeptides of the invention include those that are at least about 50% identical, at least 60% identical, at least 65% identical, more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical, to the polypeptide(s) encoded by the three reading frames of a polynucleotide comprising a nucleotide sequence of αttBl having a nucleic acid sequence as set forth in Figure 9 (or a nucleic acid sequence complementary thereto), to a polypeptide encoded by a polynucleotide contained in the deposited cDNA clones described herein, or to a polypeptide encoded by a polynucleotide hybridizing under stringent conditions to a polynucleotide comprising a nucleotide sequence of αttBl having a nucleic acid sequence as set forth in Figure 9 (or a nucleic acid sequence complementary thereto). Analogous polypeptides may be prepared that are at least about 65% identical, more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical, to the αttB2, αttPl, αttP2, αttLl, αttL2, αttRl and αttR2 polypeptides of the invention as depicted in Figure 9. The present polypeptides also include portions or fragments of the above-described polypeptides with at least 5,10, 15, 20, or 25 amino acids. By a polypeptide having an amino acid sequence at least, for example, 65% "identical" to a reference amino acid sequence of a given polypeptide of the invention is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to 35 amino acid alterations per each 100 amino acids of the reference amino acid sequence of a given polypeptide of the invention. In other words, to obtain a polypeptide having an amino acid sequence at least 65% identical to a reference amino acid sequence, up to 35% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 35% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino (N-) or carboxy (C-) terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether a given amino acid sequence is, for example, at least 65% identical to the amino acid sequence of a given polypeptide of the invention can be determined conventionally using known computer programs such as those described above for nucleic acid sequence identity determinations, or more preferably using the CLUSTAL W program (Thompson, J D , etal, Nucleic Acids Res. 22 4673-4680 (1994)) The polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art In addition, as described in detail below, the polypeptides of the present invention can be used to raise polyclonal and monoclonal antibodies which are useful in a variety of assays for detecting protein expression, localization, detection of interactions with other molecules, or for the isolation of a polypeptide (including a fusion polypeptide) of the invention In another aspect, the present invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention, which may be used to raise antibodies, particularly monoclonal antibodies, that bind specifically to a one or more of the polypeptides of the invention The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen These immunogenic epitopes are believed to be confined to a few loci on the molecule On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope " The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes (see, e.g., Geysen et al, Proc. Natl. Acad. Sci. USA 81 3998- 4002 (1983)) As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e , that contain a region of a protein molecule to which an antibody can bind), it is well-known in the art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein (see, e.g., Sutcliffe, J G , et al, Science 219:660-666 (1983)) Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are not confined to the immunodominant regions of intact proteins (i.e., immunogenic epitopes) or to the amino or carboxy termini Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein, longer peptides, especially those containing proline residues, usually are effective (Sutcliffe, J G , et al, Science 219:660-666 (1983)) Epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least five, more preferably at least seven or more amino acids contained within the amino acid sequence of a polypeptide of the invention However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a given polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided), sequences containing proline residues are particularly preferred Non-limiting examples of epitope-bearing polypeptides or peptides that can be used to generate antibodies specific for the polypeptides of the invention include certain epitope-bearing regions of the polypeptides comprising amino acid sequences encoded by polynucleotides comprising one or more of the recombination site-encoding nucleic acid molecules of the invention, including those encoding αttBl, αttB2, αttPl, αttP2, αttLl, αttL2, αttRl and αttR2 having the nucleotide sequences set forth in Figure 9 (or a nucleotide sequence complementary thereto), the complete amino acid sequences encoded by the three reading frames of the polynucleotides contained in the deposited clones described herein, and the amino acid sequences encoded by all reading frames of polynucleotides which hybridize under stringent hybridization conditions to polynucleotides having the nucleotide sequences encoding the recombination site sequences (or portions thereof) of the invention as set forth in Figure 9 (or a nucleic acid sequence complementary thereto) Other epitope-bearing polypeptides or peptides that may be used to generate antibodies specific for the polypeptides of the invention will be apparent to one of ordinary skill in the art based on the primary amino acid sequences of the polypeptides of the invention described herein, via the construction of Kyte-Doolittle hydrophilicity and Jameson-Wolf antigenic index plots of the polypeptides of the invention using, for example, PROTEAN computer software (DNASTAR, Inc , Madison, Wisconsin) The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis (see, e.g., U S Patent No 4,631,211 and Houghten, R A , Proc. Natl. Acad. Sci. USA 82 5131-5135 (1985), both of which are incorporated by reference herein in their entireties) As one of skill in the art will appreciate, the polypeptides of the present invention and epitope-bearing fragments thereof may be immobilized onto a solid support, by techniques that are well-known and routine in the art By "solid support" is intended any solid support to which a peptide can be immobilized Such solid supports include, but are not limited to nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, beads and microtitre plates Linkage of the peptide of the invention to a solid support can be accomplished by attaching one or both ends of the peptide to the support Attachment may also be made at one or more internal sites in the peptide Multiple attachments (both internal and at the ends of the peptide) may also be used according to the invention Attachment can be via an amino acid linkage group such as a primary amino group, a carboxyl group, or a sulfhydryl (SH) group or by chemical linkage groups such as with cyanogen bromide (CNBr) linkage through a spacer For non-covalent attachments to the support, addition of an affinity tag sequence to the peptide can be used such as GST (Smith, D B , and Johnson, K S , Gene 67 31 (1988)), polyhistidines (Hochuli, E , et al, J. Chromatog. 411.11 (1987)), or biotin Such affinity tags may be used for the reversible attachment of the peptide to the support Such immobilized polypeptides or fragments may be useful, for example, in isolating antibodies directed against one or more of the polypeptides of the invention, or other proteins or peptides that recognize other proteins or peptides that bind to one or more of the polypeptides of the invention, as described below As one of skill in the art will also appreciate, the polypeptides of the present invention and the epitope-bearing fragments thereof described herein can be combined with one or more fusion partner proteins or peptides, or portions thereof, including but not limited to GST, His 6 , Trx, and portions of the constant domain of immunoglobulins (Ig), resulting in chimeric or fusion polypeptides These fusion polypeptides facilitate purification of the polypeptides of the invention (EP 0 394 827, Traunecker et al, Nature 331 84- 86 (1988)) for use in analytical or diagnostic (including high-throughput) format Antibodies In another aspect, the invention relates to antibodies that recognize and bind to the polypeptides (or epitope-bearing fragments thereof) or nucleic acid molecules (or portions thereof) of the invention In a related aspect, the invention relates to antibodies that recognize and bind to one or more polypeptides encoded by all reading frames of one or more recombination site nucleic acid sequences or portions thereof, or to one or more nucleic acid molecules comprising one or more recombination site nucleic acid sequences or portions thereof, including but not limited to att sites (including αttB 1 , αttB2, αttP 1 , αttP2, αttL 1 , αttL2, αttR 1 , αttR2 and the like), lox sites (e.g., loxP, loxP511, and the like), FRT, and the like, or mutants, fragments, variants and derivatives thereof See generally U S Patent No 5,888,732, which is incorporated herein by reference in its entirety The antibodies of the present invention may be polyclonal or monoclonal, and may be prepared by any of a variety of methods and in a variety of species according to methods that are well-known in the art See, for instance, U S Patent No 5,587,287, Sutcliffe, l G , etal, Science 219:660-666 (1983), Wilson etal, Cell 37 767 (1984), and Bittle, F J , et al, J. Gen. Virol 66 2341-2354 (1985) Antibodies specific for any of the polypeptides or nucleic acid molecules described herein, such as antibodies specifically binding to one or more of the polypeptides encoded by the recombination site nucleotide sequences, or one or more nucleic acid molecules, described herein or contained in the deposited clones, antibodies against fusion polypeptides (e.g., binding to fusion polypeptides between one or more of the fusion partner proteins and one or more of the recombination site polypeptides of the invention, as described herein), and the like, can be raised against the intact polypeptides or polynucleotides of the invention or one or more antigenic polypeptide fragments thereof As used herein, the term "antibody" (Ab) may be used interchangeably with the terms "polyclonal antibody" or "monoclonal antibody" (mAb), except in specific contexts as described below These terms, as used herein, are meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab') 2 fragments) which are capable of specifically binding to a polypeptide or nucleic acid molecule of the invention or a portion thereof It will therefore be appreciated that, in addition to the intact antibodies of the invention, Fab, F(ab') 2 and other fragments of the antibodies described herein, and other peptides and peptide fragments that bind one or more polypeptides or polynucleotides of the invention, are also encompassed within the scope of the invention Such antibody fragments are typically produced by proteolytic cleavage of intact antibodies, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments) Antibody fragments, and peptides or peptide fragments, may also be produced through the application of recombinant DNA technology or through synthetic chemistry Epitope-bearing peptides and polypeptides, and nucleic acid molecules or portions thereof, of the invention may be used to induce antibodies according to methods well known in the art, as generally described herein (see, e.g., Sutcliffe, et al, supra, Wilson, et al, supra, and Bittle, F J , et al, J. Gen. Virol 66 2347-2354 (1985)) Polyclonal antibodies according to this aspect of the invention may be made by immunizing an animal with one or more of the polypeptides or nucleic acid molecules of the invention described herein or portions thereof according to standard techniques (see, e.g., Harlow, E , and Lane, D , Antibodies: A Laboratory Manual, Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press (1988), Kaufman, P B , et al, In Handbook of Molecular and Cellular Methods in Biology and Medicine, Boca Raton, Florida CRC Press, pp 468-469 (1995)) For producing antibodies that recognize and bind to the polypeptides or nucleic acid molecules of the invention or portions thereof, animals may be immunized with free peptide or free nucleic acid molecules, however, antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as albumin, KLH, or tetanus toxoid (particularly for producing antibodies against the nucleic acid molecules of the invention or portions thereof, see Harlow and Lane, supra, at page 154), or to a solid phase carrier such as a latex or glass microbead For instance, peptides containing cysteine may be coupled to carrier using a linker such as m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde Animals such as rabbits, rats and mice may be immunized with either free (if the polypeptide immunogen is larger than about 25 amino acids in length) or carrier-coupled peptides or nucleic acid molecules, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg peptide, polynucleotide, or carrier protein, and Freund's adjuvant Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of antibody which can be detected, for example, by ELISA assay using free peptide or nucleic acid molecule adsorbed to a solid surface In another approach, cells expressing one or more of the polypeptides or polynucleotides of the invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies, according to routine immunological methods In yet another method, a preparation of one or more of the polypeptides or polynucleotides of the invention is prepared and purified as described herein, to render it substantially free of natural contaminants Such a preparation may then be introduced into an animal in order to produce polyclonal antisera of greater specific activity The titer of antibodies in serum from an immunized animal, regardless of the method of immunization used, may be increased by selection of anti-peptide or anti-polynucleotide antibodies, for instance, by adsorption to the peptide or polynucleotide on a solid support and elution of the selected antibodies according to methods well known in the art In an alternative method, the antibodies of the present invention are monoclonal antibodies (or fragments thereof which bind to one or more of the polypeptides of the invention) Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al, Nature 256 495 (1975), Kohler et al, Eur. J. Immunol. 6 51 1 (1976), Kohler et al, Eur. J. Immunol. 6 292 (1976), Hammerling et al. , In Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N Y , pp 563-681 (1981)) In general, such procedures involve immunizing an animal (preferably a mouse) with a polypeptide or polynucleotide of the invention (or a fragment thereof), or with a cell expressing a polypeptide or polynucleotide of the invention (or a fragment thereof) The splenocytes of such mice are extracted and fused with a suitable myeloma cell line Any suitable myeloma cell line may be employed in accordance with the present invention, however, it is preferable to employ the parent myeloma cell line (SP 2 O), available from the American Type Culture Collection, Rockville, Maryland After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterol 80 225-232 (1981)) The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding one or more of the polypeptides or nucleic acid molecules of the invention, or fragments thereof Hence, the present invention also provides hybridoma cells and cell lines producing monoclonal antibodies of the invention, particularly that recognize and bind to one or more of the polypeptides or nucleic acid molecules of the invention Alternatively, additional antibodies capable of binding to one or more of the polypeptides of the invention, or fragments thereof, may be produced in a two-step procedure through the use of anti-idiotypic antibodies Such a method makes use of the fact that antibodies are themselves antigens, and that, therefore, it is possible to obtain an antibody which binds to a second antibody In accordance with this method, antibodies specific for one or more of the polypeptides or polynucleotides of the invention, prepared as described above, are used to immunize an animal, preferably a mouse The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to an antibody specific for one or more of the polypeptides or polynucleotides of the invention can be blocked by polypeptides of the invention themselves Such antibodies comprise anti-idiotypic antibodies to the antibodies recognizing one or more of the polypeptides or polynucleotides of the invention, and can be used to immunize an animal to induce formation of further antibodies specific for one or more of the polypeptides or polynucleotides of the invention For use, the antibodies of the invention may optionally be detectably labeled by covalent or non-covalent attachment of one or more labels, including but not limited to chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, or nuclear magnetic resonance contrast agents or other labels Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase Examples of suitable radioisotopic labels include 3 H, 11! In, 125 I, 131 I, 32 P, 35 S, 14 C, 51 Cr, "To, 58 Co, 59 Fe, 75 Se, 152 Eu, 90 Y, 67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, etc lu In is a preferred isotope where in vivo imaging is used since its avoids the problem of dehalogenation of the 125 I or I31 I-labeled monoclonal antibody by the liver In addition, this radionucleotide has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl. Med. 70 296-301 (1985), Carasquillo etal, J. Nucl. Med. 25.281-287 (1987)) For example, m In coupled to monoclonal antibodies with 1 -(P-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumorous tissues, particularly the liver, and therefore enhances specificity of tumor localization (Estebanet al., J Nucl Med 28 861-870(1987)) Examples of suitable non-radioactive isotopic labels include 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Fe Examples of suitable fluorescent labels include an 152 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, a green fluorescent protein (GFP) label, and a fluorescamine label Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label Examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and iron Typical techniques for binding the above-described labels to the antibodies of the invention are provided by Kennedy et al, Clin. Chim. Acta 70 1-31 (1976), and Schurs et al, Clin. Chim. Acta 81 1-40 (1977) Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated by reference herein It will be appreciated by one of ordinary skill that the antibodies of the present invention may alternatively be coupled to a solid support, to facilitate, for example, chromatographic and other immunological procedures using such solid phase-immobilized antibodies Included among such procedures are the use of the antibodies of the invention to isolate or purify polypeptides comprising one or more epitopes encoded by the nucleic acid molecules of the invention (which may be fusion polypeptides or other polypeptides of the invention described herein), or to isolate or purify polynucleotides comprising one or more recombination site sequences of the invention or portions thereof Methods for isolation and purification of polypeptides (and, by analogy, polynucleotides) by affinity chromatography, for example using the antibodies of the invention coupled to a solid phase support, are well-known in the art and will be familiar to one of ordinary skill The antibodies of the invention may also be used in other applications, for example to cross-link or couple two or more proteins, polypeptides, polynucleotides, or portions thereof into a structural and/or functional complex In one such use, an antibody of the invention may have two or more distinct epitope-binding regions that may bind, for example, a first polypeptide (which may be a polypeptide of the invention) at one epitope-binding region on the antibody and a second polypeptide (which may be a polypeptide of the invention) at a second epitope-binding region on the antibody, thereby bringing the first and second polypeptides into close proximity to each other such that the first and second polypeptides are able to interact structurally and/or functionally (as, for example, linking an enzyme and its substrate to carry out enzymatic catalysis, or linking an effector molecule and its receptor to carry out or induce a specific binding of the effector molecule to the receptor or a response to the effector molecule mediated by the receptor) Additional applications for the antibodies of the invention include, for example, the preparation of large-scale arrays of the antibodies, polypeptides, or nucleic acid molecules of the invention, or portions thereof, on a solid support, for example to facilitate high-throughput screening of protein or RNA expression by host cells containing nucleic acid molecules of the invention (known in the art as "chip array" protocols, see, e.g., U S Patent Nos 5,856, 101, 5,837,832, 5,770,456, 5,744,305, 5,631,734, and 5,593,839, which are directed to production and use of chip arrays of polypeptides (including antibodies) and polynucleotides, and the disclosures of which are incorporated herein by reference in their entireties) By "solid support" is intended any solid support to which an antibody can be immobilized Such solid supports include, but are not limited to nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polycarbonate, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, beads and microtitre plates Preferred are beads made of glass, latex or a magnetic material Linkage of an antibody of the invention to a solid support can be accomplished by attaching one or both ends of the antibody to the support Attachment may also be made at one or more internal sites in the antibody Multiple attachments (both internal and at the ends of the antibody) may also be used according to the invention Attachment can be via an amino acid linkage group such as a primary amino group, a carboxyl group, or a sulfhydryl (SH) group or by chemical linkage groups such as with cyanogen bromide (CNBr) linkage through a spacer For non-covalent attachments, addition of an affinity tag sequence to the peptide can be used such as GST (Smith, D B , and Johnson, K S , Gene 67 31 (1988)), polyhistidines (Hochuli, E , et al, J. Chromatog. 411 11 (1987)), or biotin Alternatively, attachment can be accomplished using a ligand which binds the Fc region of the antibodies of the invention, e.g., protein A or protein G Such affinity tags may be used for the reversible attachment of the antibodies to the support Peptides may also be recognized via specific ligand-receptor interactions or using phage display methodologies that will be familiar to the skilled artisan, for their ability to bind polypeptides of the invention or fragments thereof Kits In another aspect, the invention provides kits which may be used in producing the nucleic acid molecules, polypeptides, vectors, host cells, and antibodies, and in the recombinational cloning methods, of the invention Kits according to this aspect of the invention may comprise one or more containers, which may contain one or more of the nucleic acid molecules, primers, polypeptides, vectors, host cells, or antibodies of the invention In particular, a kit of the invention may comprise one or more components (or combinations thereof) selected from the group consisting of one or more recombination proteins (e.g. , Int) or auxiliary factors (e.g. IHF and/or Xis) or combinations thereof, one or more compositions comprising one or more recombination proteins or auxiliary factors or combinations thereof (for example, GATEWAY™ LR Clonase™ Enzyme Mix or GATEWAY™ BP Clonase™ Enzyme Mix) one or more Destination Vector molecules (including those described herein), one or more Entry Clone or Entry Vector molecules (including those described herein), one or more primer nucleic acid molecules (particularly those described herein), one or more host cells (e g competent cells, such as E. coli cells, yeast cells, animal cells (including mammalian cells, insect cells, nematode cells, avian cells, fish cells, etc ), plant cells, and most particularly E. coli DB3, DB3 1 (preferably E. coli LIBRARY EFFICIENCY® DB3 1™ Competent Cells, Life Technologies, Inc , Rockville, MD), DB4 and DB5, see U S Provisional Application No 60/122,392, filed on March 2, 1999, and the corresponding U S Utility Application No of Hartley et al. , entitled "Cells Resistant to Toxic Genes and Uses Thereof," filed on even day herewith, the disclosures of which are incorporated by reference herein in its entirety), and the like In related aspects, the kits of the invention may comprise one or more nucleic acid molecules encoding one or more recombination sites or portions thereof, such as one or more nucleic acid molecules comprising a nucleotide sequence encoding the one or more recombination sites (or portions thereof) of the invention, and particularly one or more of the nucleic acid molecules contained in the deposited clones described herein Kits according to this aspect of the invention may also comprise one or more isolated nucleic acid molecules of the invention, one or more vectors of the invention, one or more primer nucleic acid molecules of the invention, and/or one or more antibodies of the invention The kits of the invention may further comprise one or more additional containers containing one or more additional components useful in combination with the nucleic acid molecules, polypeptides, vectors, host cells, or antibodies of the invention, such as one or more buffers, one or more detergents, one or more polypeptides having nucleic acid polymerase activity, one or more polypeptides having reverse transcriptase activity, one or more transfection reagents, one or more nucleotides, and the like Such kits may be used in any process advantageously using the nucleic acid molecules, primers, vectors, host cells, polypeptides, antibodies and other compositions of the invention, for example in methods of synthesizing nucleic acid molecules (e.g. , via amplification such as via PCR), in methods of cloning nucleic acid molecules (preferably via recombinational cloning as described herein), and the like Optimization of Recombinational Cloning System The usefulness of a particular nucleic acid molecule, or vector comprising a nucleic acid molecule, of the invention in methods of recombinational cloning may be determined by any one of a number of assay methods For example, Entry and Destination vectors of the present invention may be assessed for their ability to function (i.e., to mediate the transfer of a nucleic acid molecule, DNA segment, gene, cDNA molecule or library from a cloning vector to an Expression Vector) by carrying out a recombinational cloning reaction as described in more detail in the Examples below and as described in U S Application Nos 08/663,002, filed June 7, 1996 (now U.S. Patent No. 5,888,732), 09/005,476, filed January 12, 1998, 09/177,387, filed October 23, 1998, and 60/108,324, filed November 13, 1998, the disclosures of which are incorporated by reference herein in their entireties. Alternatively, the functionality of Entry and Destination Vectors prepared according to the invention may be assessed by examining the ability of these vectors to recombine and create cointegrate molecules, or to transfer a nucleic acid molecule of interest, using an assay such as that described in detail below in Example 19. Analogously, the formulation of compositions comprising one or more recombination proteins or combinations thereof, for example GATEWAY™ LR Clonase™ Enzyme Mix and GATEWAY™ BP Clonase™ Enzyme Mix, may be optimized using assays such as those described below in Example 18. Uses There are a number of applications for the compositions, methods and kits of the present invention. These uses include, but are not limited to, changing vectors, targeting gene products to intracellular locations, cleaving fusion tags from desired proteins, operably linking nucleic acid molecules of interest to regulatory genetic sequences (e.g. , promoters, enhancers, and the like), constructing genes for fusion proteins, changing copy number, changing replicons, cloning into phages, and cloning, e.g. , PCR products, genomic DNAs, and cDNAs. In addition, the nucleic acid molecules, vectors, and host cells of the invention may be used in the production of polypeptides encoded by the nucleic acid molecules, in the production of antibodies directed against such polypeptides, in recombinational cloning of desired nucleic acid sequences, and in other applications that may be enhanced or facilitated by the use of the nucleic acid molecules, vectors, and host cells of the invention. In particular, the nucleic acid molecules, vectors, host cells, polypeptides, antibodies, and kits of the invention may be used in methods of transferring one or more desired nucleic acid molecules or DNA segments, for example one or more genes, cDNA molecules or cDNA libraries, into a cloning or Expression Vector for use in transforming additional host cells for use in cloning or amplification of, or expression of the polypeptide encoded by, the desired nucleic acid molecule or DNA segment Such recombinational cloning methods which may advantageously use the nucleic acid molecules, vectors, and host cells of the invention, are described in detail in the Examples below, and in commonly owned U S Application Nos 08/486,139, filed June 7, 1995, 08/663,002, filed June 7, 1996 (now U S Patent No 5,888,732), 09/005,476, filed January 12, 1998, 09/177,387, filed October 23, 1998, and 60/108,324. filed November 13, 1998, the disclosures of all of which are incorporated by reference herein in their entireties It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are readily apparent from the description of the invention contained herein in view of information known to the ordinarily skilled artisan, and may be made without departing from the scope of the invention or any embodiment thereof Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention Examples Example 1: Recombination Reactions of Bacteriophage λ The E. coli bacteriophage A can grow as a lytic phage, in which case the host cell is lysed, with the release of progeny virus Alternatively, lambda can integrate into the genome of its host by a process called lysogenization (see Figure 60) In this lysogenic state, the phage genome can be transmitted to daughter cells for many generations, until conditions arise that trigger its excision from the genome At this point, the virus enters the lytic part of its life cycle The control of the switch between the lytic and lysogenic pathways is one of the best understood processes in molecular biology (M Ptashne, Genetic Switch, Cell Press, 1992) The integrative and excisive recombination reactions of A, performed in vitro, are the basis of Recombinational Cloning System of the present invention They can be represented schematically as follows attB x attP <-* ■ attL x attR (where "x" signifies recombination) The four att sites contain binding sites for the proteins that mediate the reactions The wild type attP, attB, attL, and attR sites contain about 243, 25, 100, and 168 base pairs, respectively The attB x attP reaction (hereinafter referred to as a "BP Reaction," or alternatively and equivalently as an "Entry Reaction" or a "Gateward Reaction") is mediated by the proteins Int and IHF The attL x attR reaction (hereinafter referred to as an "LR Reaction," or alternatively and equivalently as a "Destination Reaction") is mediated by the proteins Int, IHF, and Xis Int (integrase) and Xis (excisionase) are encoded by the λ genome, while IHF (integration host factor) is an E. coli protein For a general review of lambda recombination, see A Landy, Ann. Rev. Biochem. 58 913-949 (1989) Example 2: Recombination Reactions of the Recombinational Cloning System The LR Reaction — the exchange of a DNA segment from an Entry Clone to a Destination Vector — is the in vitro version of the A excision reaction attL x attR =» attB + attP There is a practical imperative for this configuration after an LR Reaction in one configuration of the present method, an att site usually separates a functional motif (such as a promoter or a fusion tag) from a nucleic acid molecule of interest in an Expression Clone, and the 25 bp attB site is much smaller than the attP, attL, and attR sites Note that the recombination reaction is conservative, i e , there is no net synthesis or loss of base pairs The DNA segments that flank the recombination sites are merely switched The wild type λ recombination sites are modified for purposes of the GATEWAY™ Cloning System, as follows To create certain preferred Destination Vectors, a part (43 bp) of attR was removed, to make the excisive reaction irreversible and more efficient (W Bushman et al , Science 230 906, 1985) The attR sites in preferred Destination Vectors of the invention are 125 bp in length Mutations were made to the core regions of the att sites, for two reasons (1) to eliminate stop codons, and (2) to ensure specificity of the recombination reactions (i e , attRl reacts only with attLl, attR2 reacts only with attL2, etc ) Other mutations were introduced into the short (5 bp) regions flanking the 15 bp core regions of the attB sites to minimize secondary structure formation in single-stranded forms of attB plasmids, e g , in phagemid ssDNA or in mRNA Sequences of attB 1 and attB2 to the left and right of a nucleic acid molecule of interest after it has been cloned into a Destination Vector are given in Figure 6 Figure 61 illustrates how an Entry Clone and a Destination Vector recombine in the LR Reaction to form a co-integrate, which resolves through a second reaction into two daughter molecules The two daughter molecules have the same general structure regardless of which pair of sites, attLl and attRl or attL2 and attR2, react first to form the co-integrate The segments change partners by these reactions, regardless of whether the parental molecules are both circular, one is circular and one is linear, or both are linear In this example, selection for ampicillin resistance carried on the Destination Vector, which also carries the death gene ccdB, provides the means for selecting only for the desired attB product plasmid Example 3: Protein Expression in the Recombinational Cloning System Proteins are expressed in vivo as a result of two processes, transcription (DNA into RNA), and translation (RNA into protein) For a review of protein expression in prokaryotes and eukaryotes, see Example 13 below Many vectors (pUC, BlueScript, pGem) use interruption of a transcribed lacZ gene for blue- white screening These plasmids, and many Expression Vectors, use the lac promoter to control expression of cloned genes Transcription from the lac promoter is turned on by adding the inducer IPTG However, a low level of RNA is made in the absence of inducer, i e , the lac promoter is never completely off The result of this "leakiness" is that genes whose expression is harmful to E. coh may prove difficult or impossible to clone in vectors that contain the lac promoter, or they may be cloned only as inactive mutants In contrast to other gene expression systems, nucleic acid molecules cloned into an Entry Vector may be designed not to be expressed The presence of the strong transcriptional terminator rrnB (Orosz, et al , Ewr. J. Biochem. 201 653, 1991) just upstream of the attLl site keeps transcription from the vector promoters (drug resistance and replication origin) from reaching the cloned gene However, if a toxic gene is cloned into a Destination Vector, the host may be sick, just as in other expression systems But the reliability of subcloning by in vitro recombination makes it easier to recognize that this has happened — and easier to try another expression option in accordance with the methods of the invention, if necessary Example 4: Choosing the Right Entry Vector There are two kinds of choices that must be made in choosing the best Entry Vector, dictated by (1) the particular DNA segment that is to be cloned, and (2) what is to be accomplished with the cloned DNA segment These factors are critical in the choice of Entry Vector used, because when the desired nucleic acid molecule of interest is moved from the Entry Vector to a Destination Vector, all the base pairs between the nucleic acid molecule of interest and the Int cutting sites in attLl and attL2 (such as in Figure 6) move into the Destination Vector as well For genomic DNAs that are not expressed as a result of moving into a Destination Vector, these decisions are not as critical For example, if an Entry Vector with certain translation start signals is used, those sequences will be translated into amino acids if an amino-terminal fusion to the desired nucleic acid molecule of interest is made Whether the desired nucleic acid molecule of interest is to be expressed as fusion protein, native protein, or both, dictates whether translational start sequences must be included between the attB sites of the clone (native protein) or, alternatively, supplied by the Destination Vector (fusion protein) In particular, Entry Clones that include translational start sequences may prove less suitable for making fusion proteins, as internal initiation of translation at these sites can decrease the yield of N-terminal fusion protein These two types of expression afforded by the compositions and methods of the invention are illustrated in Figure 62 No Entry Vector is likely to be optimal for all applications The nucleic acid molecule of interest may be cloned into any of several optimal Entry Vectors As an example, consider pENTR7 (Figure 16) and pENTRl l (Figure 20), which are useful in a variety of applications, including (but not limited to) •Cloning cDNAs from most of the commercially available libraries The sites to the left and right of the ccdB death gene have been chosen so that directional cloning is possible if the DNA to be cloned does not have two or more of these restriction sites •Cloning of genes directionally Sail, BamUl, Xmnl (blunt), or Kpnl on the left of ccdB, Notl, Xhol, Xbal, or EcoRV (blunt), on the right •Cloning of genes or gene fragments with a blunt amino end at t e Xmnl site The Xmnl site has four of the six most favored bases for eukaryotic expression (see Example 13, below), so that if the first three bases of the DNA to be cloned are ATG, the open reading frame (ORF) will be expressed in eukaryotic cells (e.g., mammalian cells, insect cells, yeast cells) when it is transcribed in the appropriate Destination Vector In addition, in pENTRl 1 , a Shine-Dalgarno sequence is situated 8 bp upstream, for initiating protein synthesis in a prokaryotic host cell (particularly a bacterial cell, such as E. coli) at an ATG •Cleaving off amino terminal fusions (e.g , His 6 , GST, or thioredoxin) using the highly specific TEV (Tobacco Etch Virus) protease (available from Life Technologies, Inc ) If the nucleic acid molecule of interest is cloned at the blunt Xmnl site, TEV cleavage will leave two amino acids on the amino end of the expressed protein • Selecting against uncut or singly cut Entry Vector molecules during cloning with restriction enzymes and ligase If the ccdB gene is not removed with a double digest, it will kill any recipient E. coli cell that does not contain a mutation that makes the cell resistant to ccdB (see U S Provisional Application No 60/122,392, filed on March 2, 1999, the disclosure of which is incorporated by reference herein in its entirety) •Allowing production of amino fusions with ORFs in all cloning sites There are no stop codons (in the attLl reading frame) upstream of the ccdB gene In addition, pENTRl 1 is also useful in the following applications •Cloning cDNAs that have an Ncol site at the initiating NTG into the Ncol site Similar to the Xmnl site, this site has four of the six most favored bases for eukaryotic expression Also, a Shine-Dalgarno sequence is situated 8 bp upstream, for initiating protein synthesis in a prokaryotic host cell (particularly a bacterial cell, such as E. coli) at an ATG •Producing carboxy fusion proteins with ORFs positioned in phase with the reading frame convention for carboxy-terminal fusions (see Figure 20A) Table 1 lists some non-limiting examples of Entry Vectors and their characteristics, and Figures 10-20 show their cloning sites All of the Entry Vectors listed in Table 1 are available commercially from Life Technologies, Inc , Rockville, Maryland Other Entry Vectors not specifically listed here, which comprise alternative or additional features may be made by one of ordinary skill using routine methods of molecular and cellular biology, in view of the disclosure contained herein Entry vectors pENTRIA (Figures lOAand 10B), pENTR2B (Figures 11 A and 1 IB), and pENTR3C (Figures 12A and 12B) are almost identical, except that the restriction sites are in different reading frames. Entry vectors pENTR4 (Figures 13 A and 13B), pENTR5 (Figures 14A and 14B), and pENTR6 (Figures 15 A and 15B) are essentially identical to pENTRIA, except that the blunt Drαl site has been replaced with sites containing the ATG methionine codon: Ncol in pEΝTR4, Ndel in pENTR5, and Sphl in pENTRό. Nucleic acid molecules that contain one of these sites at the initiating ATG can be conveniently cloned in these Entry vectors. The Ncol site in pEΝTR4 is especially useful for expression of nucleic acid molecules in eukaryotic cells, since it contains many of the bases that give efficient translation (see Example 13, below). (Nucleic acid molecules of interest cloned into the Ndel site of pENTR5 are not expected to be highly expressed in eukaryotic cells, because the cytosine at position -3 from the initiating ATG is rare in eukaryotic genes.) Entry vectors pENTR7 (Figures 16A and 16B), pENTR8 (Figures 17A and 17B), and pENTR9 (Figures 18A and 18B) contain the recognition site for the TEV protease between the attLl site and the cloning sites. Cleavage sites for Xmnl (blunt), Ncol, and Ndel, respectively, are the most 5' sites in these Entry vectors. Amino fusions can be removed efficiently if nucleic acid molecules are cloned into these Entry vectors. TEV protease is highly active and highly specific. Example 5: Controlling Reading Frame One of the trickiest tasks in expression of cloned nucleic acid molecules is making sure the reading frame is correct. (Reading frame is important if fusions are being made between two ORFs, for example between a nucleic acid molecule of interest and a His6 or GST domain.) For purposes of the present invention, the following convention has been adopted: The reading frame of the DΝA cloned into any Entry Vector must be in phase with that of the attBl site shown in Figure 16A, pEΝTR7. Notice that the six As of the attLl site are split into two lysine codons (aaa aaa). The Destination Vectors that make amino fusions were constructed such that they enter the attRl site in this reading frame. Destination Vectors for carboxy terminal fusions were also constructed, including those containing His 6 (pDEST23, Figure 43), GST (pDEST24, Figure 44), or thioredoxin (pDEST25, Figure 45) C-terminal fusion sequences Therefore, if a nucleic acid molecule of interest is cloned into an Entry Vector so that the aaa aaa reading frame within the attLl site is in phase with the nucleic acid molecule's ORF, amino terminal fusions will automatically be correctly phased, for all the fusion tags This is a significant improvement over the usual case, where each different vector can have different restriction sites and different reading frames See Example 15 for a practical example of how to choose the most appropriate combinations of Entry Vector and Destination Vector Materials Unless otherwise indicated, the following materials were used in the remaining Examples included herein 5X LR Reaction Buffer: 200-250 mM (preferably 250 mM) Tris-HCl, pH 7 5 250-350 mM (preferably 320 mM) NaCl 1.25-5 mM (preferably 4 75 mM) EDTA 12 5-35 mM (preferably 22-35 mM, and most preferably 35 mM) Spermidine-HCl 1 mg/ml bovine serum albumin GATEWAY™ LR Clonase™ Enzyme Mix: per 4 μl of IX LR Reaction Buffer 150 ng carboxy-His6-tagged Int (see U S Appl Nos 60/108,324, filed November 13, 1998, and 09/438,358, filed November 12, 1999, both entirely incorporated by reference herein) 25 ng carboxy-His6-tagged Xis (see U.S. Appl. Nos. 60/108,324, filed November 13, 1998, and 09/438,358, filed November 12, 1999, both entirely incorporated by reference herein) 30 ng IHF 50% glycerol 5X BP Reaction Buffer: 125 mM Tris-HCl, pH 7.5 1 10 mM NaCl 25 mM EDTA 25 mM Spermidine-HCl 5 mg/ml bovine serum albumin GATEWAY™ BP Clonase™ Enzyme Mix: per 4 μl of IX BP Reaction Buffer: 200 ng carboxy-Hisό-tagged Int (see U.S. Appl. Nos. 60/108,324, filed November 13, 1998, and 09/438,358, filed November 12, 1999, both entirely incorporated by reference herein) 80 ng IHF 50% glycerol 10X Clonase Stop Solution: 50 mM Tris-HCl, pH 8.0 1 mM EDTA 2 mg/ml Proteinase K Example 6: LR ("Destination ") Reaction To create a new Expression Clone containing the nucleic acid molecule of interest (and which may be introduced into a host cell, ultimately for production of the polypeptide encoded by the nucleic acid molecule), an Entry Clone or Vector containing the nucleic acid molecule of interest, prepared as described herein, is reacted with a Destination Vector In the present example, a β-Gal gene flanked by attL sites is transferred from an Entry Clone to a Destination Vector Materials needed 5 X LR Reaction buffer Destination Vector (preferably linearized), 75-150 ng/μl Entry Clone containing nucleic acid molecule of interest, 100-300 ng in < 8 μl TE buffer Positive control Entry Clone (pENTR-β-Gal) DNA (See note, below) Positive control Destination Vector, pDESTl (pTrc), 75 ng/μl GATEWAY™ LR Clonase™ Enzyme Mix (stored at - 80° C) 10X Clonase Stop solution pUC19 DNN lO pg/μl Chemically competent E. coh cells (competence > lxlO 7 CFU/μg), 400 μl LB Plates containing ampicillin (100 μg/ml) and methicillin (200 μg/ml) ± X-gal and IPTG (See below) Notes Preparation of the Entry Clone DNA Miniprep DNA that has been treated with RNase works well A reasonably accurate quantitation (±50%) of the DNA to be cloned is advised, as the GATEWAY™ reaction appears to have an optimum of about 100-300 ng of Entry Clone per 20 μl of reaction mix The positive control Entry Clone, pENTR-β-Gal, permits functional analysis of clones based on the numbers of expected blue vs white colonies on LB plates containing IPTG + Bluo-gal (or X-gal), in addition to ampicillin (100 μg/ml) and methicillin (200 μg/ml) Because β-Galactosidase is a large protein, it often yields a less prominent band than many smaller proteins do on SDS protein gels In the Positive Control Entry Vector pENTR-β-Gal, the coding sequence of β-Gal has been cloned into pENTRl l (Figures 20A and 20B), with translational start signals permitting expression in E coli, as well as in eukaryotic cells The positive control Destination Vector, for example pDESTl (Figure 21), is preferably linearized To prepare X-gal + IPTG plates, either of the following protocols may be used A With a glass rod, spread over the surface of an LB agar plate 40 μl of 20 mg/ml X-gal (or Bluo-gal) in DMF plus 4 μl 200 mg/ml IPTG Allow liquid to adsorb into agar for 3-4 hours at 37° C before plating cells B To liquid LB agar at -45 °C, add X-gal (or Bluo-Gal) (20 mg/ml in DMF) to make 50 μg/ml and IPTG (200 mM in water) to make 0 5-1 mM, just prior to pouring plates Store X-gal and Bluo-Gal in a light-shielded container Colony color may be enhanced by placing the plates at 5 °C for a few hours after the overnight incubation at 37 °C Protocol B can give more consistent colony color than A, but A is more convenient when selection plates are needed on short notice Recombination in Clonase reactions continues for many hours While incubations of 45-60 minutes are usually sufficient, reactions with large DNAs, or in which both parental DNAs are supercoiled, or which will be transformed into cells of low competence, can be improved with longer incubation times, such as 2-24 hours at 25 °C Procedure 1 Assemble reactions as follows (combine all components at room temperature, except GATEWAY™ LR Clonase™ Enzyme Mix ("Clonase LR"), before removing Clonase LR from frozen storage) 2 Remove the GATEWAY™ LR Clonase™ Enzyme Mix from the -80° C freezer, place immediately on ice The Clonase takes only a few minutes to thaw 3 Add 4 μl of GATEWAY™ LR Clonase™ Enzyme Mix to reactions #2 and #4, 4 Return GATEWAY™ LR Clonase™ Enzyme Mix to - 80° C freezer 5 Incubate tubes at 25 ° for at least 60 minutes 6 Add 2 μl Clonase Stop solution to all reactions Incubate for 20 min at 37°C (This step usually increases the total number of colonies obtained by 10-20 fold ) 7 Transform 2 μl into 100 μl competent E. cob Select on plates containing ampicillin at 100 μg/ml Example 7: Transformation ofE. coli To introduce cloning or Expression Vectors prepared using the recombinational cloning system of the invention, any standard E. cob transformation protocol should be satisfactory The following steps are recommended for best results 1 Let the mixture of competent cells and Recombinational Cloning System reaction product stand on ice at least 15 minutes prior to the heat-shock step This gives time for the recombination proteins to dissociate from the DNA, and improves the transformation efficiency 2 Expect the reaction to be about l%-5% efficient, i e , 2 μl of the reaction should contain at least 100 pg of the Expression Clone plasmid (taking into account the amounts of each parental plasmid in the reaction, and the subsequent dilution) If the E coli cells have a competence of 10 7 CFU/μg, 100 pg of the desired clone plasmid will give about 1000 colonies, or more, if the entire transformation is spread on one ampicillin plate 3 Always do a control pUC DNA transformation If the number of colonies is not what you expect, the pUC DNA transformation gives you an indication of where the problem was Example 8: Preparation of attB-PCR Product For preparation of attB-PCR products in the PCR cloning methods described in Example 9 below, PCR primers containing attB 1 and attB2 sequences are used The attB 1 and attB2 primer sequences are as follows attBl: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT- (template-specific sequence)-3 ' attB2: 5'-GGGGACCACTTTGTACANGAAAGCTGGGT-(template-specific sequence)-3 ' The attB 1 sequence should be added to the amino primer, and the attB2 sequence to the carboxy primer The 4 guanines at the 5' ends of each of these primers enhance the efficiency of the minimal 25 bp attB sequences as substrates for use in the cloning methods of the invention Standard PCR conditions may be used to prepare the PCR product The following suggested protocol employs PLATINUM Taq DNA Polymerase High Fidelity®, available commercially from Life Technologies, Inc (Rockville, MD) This enzyme mix eliminates the need for hot starts, has improved fidelity over Taq, and permits synthesis of a wide range of amplicon sizes, from 200 bp to 10 kb, or more, even on genomic templates Materials needed: •PLATINUM Taq DNA Polymerase High Fidelity® (Life Technologies, Inc ) •attBl- and attB2- containing primer pair (see above) specific for your template •DNA template (linearized plasmid or genomic DNA) • 10X High Fidelity PCR Buffer • lO mM dNTP mix •PEG/MgCl 2 Mix (30% PEG 8000, 30 mM MgCl 2 ) Procedure 1 ) Assemble the reaction as follows * Use of higher amounts of plasmid template may permit fewer cycles (10-15) of PCR 2 ) Add 2 drops mineral oil, as appropriate 3 ) Denature for 30 sec at 94 °C 4 ) Perform 25 cycles 94°C for 15 sec-30 sec 55°C for 15 sec-30 sec 68 °C for 1 min per kb of template 5 ) Following the PCR reaction, apply 1-2 μl of the reaction mixture to an agarose gel, together with size standards (e.g. , 1 Kb Plus Ladder, Life Technologies, Inc ) and quantitation standards (e.g., Low Mass Ladder, Life Technologies, Inc ), to assess the yield and uniformity of the product Purification of the PCR product is recommended, to remove attB primer dimers which can clone efficiently into the Entry Vector The following protocol is fast and will remove DNA <300 bp in size 6 ) Dilute the 50 μl PCR reaction to 200 μl with TE 7 ) Add 100 μl PEG/MgCl 2 Solution Mix and centrifuge immediately at 13,000 RPM for 10 min at room temperature Remove the supernatant (pellet is clear and hard to see) 8 ) Dissolve the pellet in 50 μl TE and check recovery on a gel If the starting PCR template is a plasmid that contains the gene for Kan r , it is advisable to treat the completed PCR reaction with the restriction enzyme Dpnl, to degrade the plasmid since unreacted residual starting plasmid is a potential source of false-positive colonies from the transformation of the GATEWAY™ Cloning System reaction Adding ~5 units of Dpnl to the completed PCR reaction and incubating for 15 min at 37°C will eliminate this potential problem Heat inactivate the Dpnl at 65 ° C for 15 min, prior to using the PCR product in the GATEWAY™ Cloning System reaction Example 9: Cloning attB-PCR products into Entry Vectors via the BP ("Gateward") Reaction The addition of 5 '-terminal attB sequences to PCR primers allows synthesis of a PCR product that is an efficient substrate for recombination with a Donor (attP) Plasmid in the presence of GATEWAY™ BP Clonase™ Enzyme Mix This reaction produces an Entry Clone of the PCR product (See Figure 8) The conditions of the Gateward Cloning reaction with an attB PCR substrate are similar to those of the BP Reaction (see Example 10 below), except that the attB-PCR product (see Example 8) substitutes for the Expression Clone, and the attB-PCR positive control (attB-te ) substitutes for the Expression Clone Positive Control (GFP) Materials needed • 5 X BP Reaction Buffer • Desired attB-PCR product DNN 50-100 ng in < 8 μl TE • Donor (attP) Plasmid (Figures 49-54), 75 ng/μl, supercoiled DΝA • attB-te PCR product positive control, 25 ng/μl • GATEWAY™ BP Clonase™ Enzyme Mix (stored at - 80° C) • lOx Clonase Stop Solution • pUC19 DΝN lO pg/μl • Chemically competent E coli cells (competence > lxl 0 7 CFU/μg), 400 μl Notes »Preparation of attB-PCR DNA see Example 8 •The Positive Control attB-te PCR product contains a functional copy of the te gene of pBR322, with its own promoter By plating the transformation of the control BP Reaction on kanamycin (50 μg/ml) plates (if kan r Donor Plasmids are used, see Figures 49-52) or an alternative selection agent (e.g., gentamycin, if gen r Donor Plasmids are used, see Figure 54), and then picking about 50 of these colonies onto plates with tetracycline (20 μg/ml), the percentage of Entry Clones containing functional tet r among the colonies from the positive control reaction can be determined (% Expression Clones = (number of te + kan r (or gen r ) colonies/kan r (or gen r ) colonies). Procedure: 1. Assemble reactions as follows. Combine all components except GATEWAY™ BP Clonase™ Enzyme Mix, before removing GATEWAY™ BP Clonase™ Enzyme Mix from frozen storage. 2. Remove the GATEWAY™ BP Clonase™ Enzyme Mix from the -80° C freezer, place immediately on ice. The Clonase takes only a few minutes to thaw. 3. Add 4 μl of GATEWAY™ BP Clonase™ Enzyme Mix to the subcloning reaction, mix. 4. Return GATEWAY™ BP Clonase™ Enzyme Mix to - 80° C freezer. 5. Incubate tubes at 25° for at least 60 minutes. 6 Add 2 μl Proteinase K (2 μg/μl) to all reactions Incubate for 20 min at 37 °C 7 Transform 2 μl into 100 μl competent E coli, as per 3 2, above Select on LB plates containing kanamycin, 50 μg/ml Results In initial experiments, primers for amplifying tetR and ampR from pBR322 were constructed containing only the tetR- or ampR-specific targeting sequences, the targeting sequences plus attB 1 (for forward primers) or attB2 (for reverse primers) sequences shown in Figure 9, or the attB l or attB2 sequences with a 5' tail of four guanines The construction of these primers is depicted in Figure 65 After PCR amplification of tetR and ampR from pBR322 using these primers and cloning the PCR products into host cells using the recombinational cloning system of the invention, the results shown in Figure 66 were obtained These results demonstrated that primers containing attB sequences provided for a somewhat higher number of colonies on the tetracycline and ampicillin plates However, inclusion of the 5' extensions of four or five guanines on the primers in addition to the attB sequences provided significantly better cloning results, as shown in Figures 66 and 67 These results indicate that the optimal primers for cloning of PCR products using recombinational cloning will contain the recombination site sequences with a 5' extension of four or five guanine bases To determine the optimal stoichiometry between attB-containing PCR products and attP-containing Donor plasmid, experiments were conducted where the amount of PCR product and Donor plasmid were varied during the BP Reaction Reaction mixtures were then transformed into host cells and plated on tetracycline plates as above Results are shown in Figure 68 These results indicate that, for optimal recombinational cloning results with a PCR product in the size range of the tet gene, the amounts of attP-containing Donor plasmids are between about 100-500 ng (most preferably about 200-300 ng), while the optimal concentrations of attB-containing PCR products is about 25-100 ng (most preferably about 100 ng), per 20 μl reaction Experiments were then conducted to examine the effect of PCR product size on efficiency of cloning via the recombinational cloning approach of the invention PCR products containing attBl and attB2 sites, at sizes 256 bp, 1 kb, 1 4 kb, 3 4 kb, 4 6 kb, 6 9 kb and 10 1 kb were prepared and cloned into Entry vectors as described above, and host cells were transformed with the Entry vectors containing the cloned PCR products For each PCR product, cloning efficiency was calculated relative to cloning of pUC19 positive control plasmids as follows CFU/ng attB PCR product Size (kb) PCR product Cloning Efficiency = X - CFU/ng pUC 19 control Size (kb) pUC 19 control The results of these experiments are depicted in Figures 69A-69C (for 256 bp PCR fragments), 70A-70C (for 1 kb PCR fragments), 71 A-71C (for 1 4 kb PCR fragments), 72A-72C (for 3 4 kb PCR fragments), 73A-73C (for 4 6 kb PCR fragments), 74 (for 6 9 kb PCR fragments), and 75-76 (for 10 1 kb PCR fragments) The results shown in these figures are summarized in Figure 77, for different weights and moles of input PCR DNA Together, these results demonstrate that attB-containing PCR products ranging in size from about 0 25 kb to about 5 kb clone relatively efficiently in the recombinational cloning system of the invention While PCR products larger than about 5 kb clone less efficiently (apparently due to slow resolution of cointegrates), longer incubation times during the recombination reaction appears to improve the efficiency of cloning of these larger PCR fragments Alternatively, it may also be possible to improve efficiency of cloning of large (> about 5 kb) PCR fragments by using lower levels of input attP Donor plasmid and perhaps attB-containing PCR product, and/or by adjusting reaction conditions (e.g. , buffer conditions) to favor more rapid resolution of the cointegrates Example 10: The BP Reaction One purpose of the Gateward ("Entry") reaction is to convert an Expression Clone into an Entry Clone This is useful when you have isolated an individual Expression Clone from an Expression Clone cDNA library, and you wish to transfer the nucleic acid molecule of interest into another Expression Vector, or to move a population of molecules from an attB or attL library Alternatively, you may have mutated an Expression Clone and now wish to transfer the mutated nucleic acid molecule of interest into one or more new Expression Vectors In both cases, it is necessary first to convert the nucleic acid molecule of interest to an Entry Clone Materials needed 5 X BP Reaction Buffer Expression Clone DNN 100-300 ng in < 8 μl TE • Donor (attP) Vector, 75 ng/μl, supercoiled DΝA Positive control attB-tet-PCR DΝN 25 ng/μl GATEWAY™ BP Clonase™ Enzyme Mix (stored at - 80 °C) Clonase Stop Solution (Proteinase K, 2 μg/μl) Notes Preparation of the Expression Clone DNA Miniprep DNA treated with RNase works well 1 As with the LR Reaction (see Example 14), the BP Reaction is strongly influenced by the topology of the reacting DNAs In general, the reaction is most efficient when one of the DNAs is linear and the other is supercoiled, compared to reactions where the DNAs are both linear or both supercoiled Further, linearizing the attB Expression Clone (anywhere within the vector) will usually give more colonies than linearizing the Donor (attP) Plasmid If finding a suitable cleavage site within your Expression Clone vector proves difficult, you may linearize the Donor (attP) Plasmid between the attPl and attP2 sites (for example, at the Ncol site), avoiding the ccdB gene Maps of Donor (attP) Plasmids are given in Figures 49-54 Procedure 1 Assemble reactions as follows Combine all components at room temperature, except GATEWAY™ BP Clonase™ Enzyme Mix, before removing GATEWAY™ BP Clonase™ Enzyme Mix from freezer 2 Remove the GATEWAY™ BP Clonase™ Enzyme Mix from the -80 °C freezer, place immediately on ice The mixture takes only a few minutes to thaw 3 Add 4 μl of GATEWAY™ BP Clonase™ Enzyme Mix to the subcloning reaction, mix 4 Return GATEWAY™ BP Clonase™ Enzyme Mix to - 80° C freezer 5 Incubate tubes at 25 ° for at least 60 minutes If both the attB and attP DNAs are supercoiled, incubation for 2-24 hours at 25 °C is recommended 6 Add 2 μl Clonase Stop Solution Incubate for 10 min at 37 °C 7 Transform 2 μl into 100 μl competent E coli, as above Select on LB plates containing 50 μg/ml kanamycin Example 11: Cloning PCR Products into Entry Vectors using Standard Cloning Methods Preparation of Entry Vectors for Cloning: of PCR Products All of the Entry Vectors of the invention contain the death gene ccdB as a stuffer between the "left" and "right" restriction sites The advantage of this arrangement is that there is virtually no background from vector that has not been cut with both restriction enzymes, because the presence of the ccdB gene will kill all standard E coli strains Thus it is necessary to cut each Entry Vector twice, to remove the ccdB fragment We strongly recommend that, after digestion of the Entry Vector with the second restriction enzyme, you treat the reaction with phosphatase (calf intestine alkaline phosphatase, CIAP or thermosensitive alkaline phosphatase, TSAP) The phosphatase can be added directly to the reaction mixture, incubated for an additional time, and inactivated This step dephosphorylates both the vector and ccdB fragments, so that during subsequent ligation there is less competition between the ccdB fragment and the DNA of interest for the termini of the Entry Vector Blunt Cloning of PCR products Generally PCR products do not have 5' phosphates (because the primers are usually 5' OH), and they are not necessarily blunt (On this latter point, see Brownstein, et al , BioTechniques 20 1006, 1996 for a discussion of how the sequence of the primers affects the addition of single 3 ' bases ) The following protocol repairs these two defects In a 0 5 ml tube, ethanol precipitate about 40 ng of PCR product (as judged from an agarose gel) 1 Dissolve the precipitated DNA in 10 μl comprising 1 μl 10 mM rATP, 1 μl mixed 2 mM dNTPs (i e , 2 mM each dATP, dCTP, dTTP, and dGTP), 2 μl 5x T4 polynucleotide kinase buffer (350 mM Tris HCl (pH7 6), 50 mM MgCl 2 , 500mM KC1, 5 mM 2-mercaptoethanol) 10 units T4 polynucleotide kinase, 1 μl T4 DNA polymerase, and water to 10 μl 2 Incubate the tube at 37° for 10 minutes, then at 65 ° for 15 minutes, cool, centrifuge briefly to bring any condensate to the tip of the tube 3 Add 5 μl of the PEG/MgCl 2 solution, mix and centrifuge at room temperature for 10 minutes Discard supernatant 4 Dissolve the invisible precipitate in 10 μl containing 2 μl 5x T4 DNA ligase buffer (Life Technologies, Inc ), 0 5 units T4 DNA ligase, and about 50 ng of blunt, phosphatase-treated Entry Vector 5 Incubate at 25 ° for 1 hour, then 65 ° for 10 minutes Add 90 μl TE, transform 10 μl into 50 - 100 μl competent E coli cells 6 Plate on kanamycin Note: In the above protocol, steps b-c simultaneously polish the ends of the PCR product (through the exonuclease and polymerase activities of T4 DNA polymerase) and phosphorylate the 5' ends (using T4 polynucleotide kinase) It is necessary to inactivate the kinase, so that the blunt, dephosphorylated vector in step e cannot self ligate Step d (the PEG precipitation) removes all small molecules (primers, nucleotides), and has also been found to improve the yield of cloned PCR product by 50 fold Cloning PCR Products after Digestion with Restriction Enzymes Efficient cloning of PCR products that have been digested with restriction enzymes includes three steps inactivation of Taq DNA polymerase, efficient restriction enzyme cutting, and removal of small DNA fragments Inactivation of Taq DNA Polymerase Carryover of Taq DNA polymerase and dNTPs into a RE digestion significantly reduces the success in cloning a PCR product (D Fox et al , FOCUS 20(1) 15, 1998), because Taq DNA polymerase can fill in sticky ends and add bases to blunt ends Either TAQQUENCH™ (obtainable from Life Technologies, Inc , Rockville, Maryland) or extraction with phenol can be used to inactivate the Taq Efficient Restriction Enzyme Cutting Extra bases on the 5' end of each PCR primer help the RE cut near ends of PCR products With the availability of cheap primers, adding 6 to 9 bases on the 5' sides of the restriction sites is a good investment to ensure that most of the ends are digested Incubation of the DNA with a 5-fold excess of restriction enzyme for an hour or more helps ensure success Removal of Small Molecules before Ligation Primers, nucleotides, primer dimers, and small fragments produced by the restriction enzyme digestion, can all inhibit or compete with the desired ligation of the PCR product to the cloning vector This protocol uses PEG precipitation to remove small molecules Protocol for cutting the ends of PCR products with restriction enzyme(s) 1 Inactivation of Taq DNA polymerase in the PCR product Option A Extraction with Phenol Al Dilute the PCR reaction to 200 μl with TE Add an equal volume of phenol chloroform isoamyl alcohol, vortex vigorously for 20 seconds, and centrifuge for 1 minute at room temperature Discard the lower phase A2 Extract the phenol from the DNA and concentrate as follows Add an equal volume of 2-butanol (colored red with "Oil Red O" from Aldrich, if desired), vortex briefly, centrifuge briefly at room temperature Discard the upper butanol phase Repeat the extraction with 2-butanol This time the volume of the lower aqueous phase should decrease significantly Discard the upper 2-butanol phase A3 Ethanol precipitate the DNA from the aqueous phase of the above extractions Dissolve in a 200 μl of a suitable restriction enzyme (RE) buffer Option B Inactivation with TaqQuench Bl Ethanol precipitate an appropriate amount of PCR product (100 ng to 1 μg), dissolve in 200 μl of a suitable RE buffer B2 Add 2 μl TaqQuench 2 Add 10 to 50 units of restriction enzyme and incubate for at least 1 hour Ethanol precipitate if necessary to change buffers for digestion at the other end of the PCR product 3 Add Vi volume of the PEG/MgCl 2 mix to the RE digestion Mix well and immediately centrifuge at room temperature for 10 minutes Discard the supernatant (pellet is usually invisible), centrifuge again for a few seconds, discard any remaining supernatant 4 Dissolve the DNA in a suitable volume of TE (depending on the amount of PCR product in the original amplification reaction) and apply an aliquot to an agarose gel to confirm recovery Apply to the same gel 20-100 ng of the appropriate Entry Vector that will be used for the cloning Example 12: Determining The Expected Size of the GATEWA Y™ Cloning Reaction Products If you have access to a software program that will electronically cut and splice sequences, you can create electronic clones to aid you in predicting the sizes and restriction patterns of GATEWAY™ Cloning System recombination products The cleavage and ligation steps performed by the enzyme Int in the GATEWAY™ Cloning System recombination reactions mimic a restriction enzyme cleavage that creates a 7-bp 5 '-end overhang followed by a ligation step that reseals the ends of the daughter molecules The recombination proteins present in the Clonase cocktails (see Example 19 below) recognize the 15 bp core sequence present within all four types of att sites (in addition to other flanking sequences characteristic of each of the different types of att sites) By treating these sites in your software program as if they were restriction sites, you can cut and splice your Entry Clones with various Destination Vectors and obtain accurate maps and sequences of the expected results from your GATEWAY™ Cloning System reactions Example 13: Protein Expression Brief Review of Protein Expression Transcription The most commonly used promoters in E. coh Expression Vectors are variants of the lac promoter, and these can be turned on by adding IPTG to the growth medium It is usually good to keep promoters off until expression is desired, so that the host cells are not made sick by the overabundance of some heterologous protein This is reasonably easy in the case of the lac promoters used in E coli One needs to supply the lac I gene (or its more productive relative, the lac I q gene) to make lac repressor protein, which binds near the promoter and keeps transcription levels low Some Destination Vectors for E. cob expression carry their own /αcl q gene for this purpose (However, lac promoters are always a little "on," even in the absence of IPTG ) Controlling transcription in eukaryotic cells is not nearly so straightforward or efficient The tetracycline system of Bujard and colleagues is the most successful approach, and one of the Destination Vectors (pDESTl 1 , Figure 31) has been constructed to supply this function Translation Ribosomes convert the information present in mRNA into protein Ribosomes scan RNA molecules looking for methionine (AUG) codons, which begin nearly all nascent proteins Ribosomes must, however, be able to distinguish between AUG codons that code for methionine in the middle of proteins from those at the start Most often ribosomes choose AUGs that are 1) first in the RNA (toward the 5' end), and 2) have the proper sequence context In E coli the favored context (first recognized by Shine and Dalgarno, Ewr. J. Biochem. 57 221 (1975)) is a run of purines (As and Gs) from five to 12 bases upstream of the initiating AUG, especially AGGAGG or some variant In eukaryotes, a survey of translated mRNAs by Kozak (J. Biol Chem. 266 19867 (1991)) has revealed a preferred sequence context, gcc Ace ATGG, around the initiating methionine, with the A at -3 being most important, and a purine at +4 (where the A of the ATG is +1), preferably a G, being next most influential Having an A at -3 is enough to make most ribosomes choose the first AUG of an mRNA, in plants, insects, yeast, and mammals (For a review of initiation of protein synthesis in eukaryotic cells, see Pain, V M Εur j Biochem 236 747-771, 1996 ) Consequences of Translation Signals for GA TEW A Y™ Cloning System First, translation signals (Shine-Dalgarno in Ε coli, Kozak in eukaryotes) have to be close to the initiating ATG The attB site is 25 base pairs long Thus if translation signals are desired near the natural ATG of the nucleic acid molecule of interest, they must be present in the Entry Clone of that nucleic acid molecule of interest Also, when a nucleic acid molecule of interest is moved from an Entry Clone to a Destination vector, any translation signals will move along The result is that the presence or absence of Shine-Dalgarno and/or Kozak sequences in the Entry Clone must be considered, with the eventual Destination Vectors to be used in mind Second, although ribosomes choose the 5 ' ATG most often, internal ATGs are also used to begin protein synthesis The better the translation context around this internal ATG, the more internal translation initiation will be seen This is important in the GATEWAY™ Cloning System, because you can make an Entry Clone of your nucleic acid molecule of interest, and arrange to have Shine- Dalgarno and/or Kozak sequences near the ATG When this cassette is recombined into a Destination Vector that transcribes your nucleic acid molecule of interest, you get native protein If you want, you can make a fusion protein in a different Destination Vector, since the Shine-Dalgarno and/or Kozak sequences do not contain any stop signals in the same reading frame However, the presence of these internal translation signals may result in a significant amount of native protein being made, contaminating, and lowering the yield of, your fusion protein This is especially likely with short fusion tags, like His6 A good compromise can be recommended If an Entry Vector like pENTR7 (Figure 16) or pENTR8 (Figure 17) is chosen, the Kozak bases are present for native eukaryotic expression The context for E coli translation is poor, so the yield of an amino-terminal fusion should be good, and the fusion protein can be digested with the TEV protease to make near-native protein following purification Recommended Conditions for Synthesis of Proteins in E. cob When making proteins inE. coh it is advisable, at least initially, to incubate your cultures at 30°C, instead of at 37°C Our experience indicates that proteins are less likely to form aggregates at 30° C In addition, the yields of proteins from cells grown at 30 °C frequently are improved The yields of proteins that are difficult to express may also be improved by inducing the cultures in mid-log phase of growth, using cultures begun in the morning from overnight growths, as opposed to harvesting directly from an overnight culture In the latter case, the cells are preferably in late log or stationary growth, which can favor the formation of insoluble aggregates Example 14: Constructing Destination Vectors from Existing Vectors Destination Vectors function because they have two recombination sites, attRl and attR2, flanking a chloramphenicol resistance (CmR) gene and a death gene, ccdB The GATEWAY™ Cloning System recombination reactions exchange the entire Cassette (except for a few bases comprising part of the attB sites) for the DNA segment of interest from the Entry Vector Because attRl, CmR, ccdB gene, and attR2 are contiguous, they can be moved on a single DNA segment If this Cassette is cloned into a plasmid, the plasmid becomes a Destination Vector Figure 63 shows a schematic of the GATEWAY™ Cloning System Cassette, attR cassettes in all three reading frames contained in vectors pEZC15101, pEZC15102 and pEZC15103 are shown in Figures 64A, 64B, and 64C, respectively The protocol for constructing a Destination Vector is presented below Keep in mind the following points • Destination Vectors must be constructed and propagated in one of the DB strains of E. cob (e.g., DB3 1, and particularly E. cob LIBRARY EFFICIENCY® DB3 1™ Competent Cells) available from Life Technologies, Inc (and described in detail in U S Provisional Application No 60/122,392, filed on March 2, 1999, which is incorporated herein by reference), because the ccdB death gene will kill any E. cob strain that has not been mutated such that it will survive the presence of the ccdB gene • If your Destination Vector will be used to make a fusion protein, a GATEWAY™ Cloning System cassette with the correct reading frame must be used The nucleotide sequences of the ends of the cassettes are shown in Figure 78 The reading frame of the fusion protein domain must be in frame with the core region of the attRl site (for an amino terminal fusion) so that the six As are translated into two lysine codons For a C-terminal fusion protein, translation through the core region of the attR2 site should be in frame with -TAC-AAA-, to yield -Tyr-Lys- • Note that each reading frame Cassette has a different unique restriction site between the chloramphenicol resistance and ccdB genes (M for reading frame A, Bglϊl for reading frame B, andNbαl for reading frame C, see Figure 63) • Most standard vectors can be converted to Destination Vectors, by inserting the Entry Cassette into the MCS of that vector Protocol for Making a Destination Vector 1 If the vector will make an amino fusion protein, it is necessary to keep the "aaa aaa" triplets in attRl in phase with the triplets of the fusion protein Determine which Entry cassette to use as follows a.) Write out the nucleotide sequence of the existing vector near the restriction site into which the Entry cassette will be cloned These must be written in triplets corresponding to the amino acid sequence of the fusion domain b.) Draw a vertical line through the sequence that corresponds to the restriction site end, after it has been cut and made blunt, i e , after filling in a protruding 5' end or polishing a protruding 3' end c.) Choose the appropriate reading frame cassette • If the coding sequence of the blunt end ends after a complete codon triplet, use the reading frame A cassette See Figures 78, 79 and 80 •If the coding sequence of the blunt end ends in a single base, use the reading frame B cassette See Figures 78, 79 and 81 •If the coding sequence of the blunt end ends in two bases, use the reading frame C cassette See Figures 78, 79, 82A-B, and 83 A-C 2 Cut one to five micrograms of the existing plasmid at the position where you wish your nucleic acid molecule of interest (flanked by att sites) to be after the recombination reactions Note it is better to remove as many of the MCS restriction sites as possible at this step This makes it more likely that restriction enzyme sites within the GATEWAY™ Cloning System Cassette will be unique in the new plasmid, which is important for linearizing the Destination Vector (Example 14, below) 3 Remove the 5' phosphates with alkaline phosphatase While this is not mandatory, it increases the probability of success 4 Make the end(s) blunt with fill-in or polishing reactions For example, to 1 μg of restriction enzyme-cut, ethanol-precipitated vector DNA, add i 20 μl 5x T4 DNA Polymerase Buffer (165 mM Tris-acetate (pH 7 9), 330 mM Na acetate, 50 mM Mg acetate, 500 μg/ml BSA, 2 5 mM DTT) ii 5 μl lOmM dNTP mix iii 1 Unit of T4 DNA Polymerase iv Water to a final volume of 100 μl v Incubate for 15 min at 37 °C 5 Remove dNTPs and small DNA fragments Ethanol precipitate (add three volumes of room temperature ethanol containing 0 1 M sodium acetate, mix well, immediately centrifuge at room temperature 5 - 10 minutes), dissolve wet precipitate in 200 μl TE, add 100 μl 30% PEG 8000, 30 mM MgCl 2 , mix well, immediately centrifuge for 10 minutes at room temperature, discard supernatant, centrifuge again a few seconds, discard any residual liquid 6 Dissolve the DNA to a final concentration of 10 - 50 ng per microliter Apply 20 - 100 ng to a gel next to supercoiled plasmid and linear size standards to confirm cutting and recovery The cutting does not have to be 100% complete, since you will be selecting for the chloramphenicol marker on the Entry cassette 7 In a 10 μl ligation reaction combine 10 - 50 ng vector, 10 - 20 ng of Entry Cassette (Figure 79), and 0 5 units T4 DNA ligase in ligase buffer After one hour (or overnight, whichever is most convenient), transform 1 μl into one of the DB strains of competent E. cob cells with a gyr A462 mutation (See U S Provisional Application No 60/122,392, filed on March 2, 1999, which is incorporated herein by reference), preferably DB3 1, and most preferably E. coh LIBRARY EFFICIENCY® DB3 1™ Competent Cells The ccdB gene on the Entry Cassette will kill other strains of E. cob that have not been mutated so as to survive the presence of the ccdB gene 8 After expression in SOC medium, plate 10 μl and 100 μl on chloramphenicol- containing (30 μg / ml) plates, incubate at 37° C 9 Pick colonies, make miniprep DNA Treat the miniprep with RNase A and store in TE Cut with the appropriate restriction enzyme to determine the orientation of the Cassette Choose clones with the attRl site next to the amino end of the protein expression function of the plasmid Notes on Using Destination Vectors • We have found that about ten-fold more colonies result from a GATEWAY™ Cloning System reaction if the Destination Vector is linear or relaxed If the competent cells you use are highly competent (>10 8 per microgram), linearizing the Destination Vector is less essential • The site or sites used for the linearization must be within the Entry Cassette Sites that cut once or twice within each cassette are shown in Figures 80-82 • Minipreps of Destination Vectors will work fine, so long as they have been treated with RNase Since most DB strains are endA- (See U S Provisional Application No 60/122,392, filed on March 2, 1999, which is incorporated herein by reference), minipreps can be digested with restriction enzymes without a prior phenol extraction • Reading the OD 260 of miniprep DNA is inaccurate unless the RNA and ribonucleotides have been removed, for example, by a PEG precipitation Example 15: Some Options in Choosing Appropriate Entry Vectors and Destination Vectors: An Example In some applications, it may be desirable to express a nucleic acid molecule of interest in two forms as an amino-terminal fusion in E. cob, and as a native protein in eukaryotic cells This may be accomplished in any of several ways Option 1: Your choices depend on your nucleic acid molecule of interest and the fragment that contains it, as well as the available Entry Vectors For eukaryotic translation, you need consensus bases according to Kozak (J. Biol. Chem. 266:19861, 1991) near the initiating methionine (ATG) codon All of the Entry Vectors offer this motif upstream of the Xmnl site (blunt cutter) One option is to amplify your nucleic acid molecule of interest, with its ATG, by PCR, making the amino end blunt and the carboxy end containing the natural stop codon followed by one of the "right side" restriction sites (EcoRI, Notl, Xhol, EcoRV, or Nbαl of the pΕΝTR vectors) If you know your nucleic acid molecule of interest does not have, for example, an Xhol site, you can make a PCR product that has this structure Xho I 5' ATG nnn nnn nnn TAA etc gag nnn nnn 3' 3' tac nnn nnn nnn att gag etc nnn nnn 5' After cutting with Xhol, the fragment is ready to clone 5 ' ATG nnn nnn nnn TAA c 3 ' 3' tac nnn nnn nnn att gag ct 5' (If you follow this example, don't forget to put a phosphate on the amino oligo ) Option 2: This PCR product could be cloned into two Entry Vectors to give the desired products, between the Xmnl and Xhol sites pENTRIA (Figures ION 10B ) or pEΝTR7 (Figures 16A, 16B) If you clone into pENTRIA, amino fusions will have the minimal number of amino acids between the fusion domain and your nucleic acid molecule of interest, but the fusion cannot be removed with TEV protease The converse is true of clones in pEΝTR7, i e , an amino fusion can be cleaved with TEV protease, at the cost of more amino acids between the fusion and your nucleic acid molecule of interest In this example, let us choose to clone our hypothetical nucleic acid molecule of interest into pENTR7, between the Xmnl and Xhol sites Once this is accomplished, several optional protocols using the Entry Clone pENTR7 may be followed Option 3: Since the nucleic acid molecule of interest has been amplified with PCR, it may be desirable to sequence it To do this, transfer the nucleic acid molecule of interest from the Entry Vector into a vector that has priming sites for the standard sequencing primers Such a vector is pDESTό (Figures 26N 26B) This Destination Vector places the nucleic acid molecule of interest in the opposite orientation to the lac promoter (which is leaky ~ see Example 3 above) If the gene product is toxic to E. cob, this Destination Vector will minimize its toxicity Option 4 : While the sequencing is going on, you might wish to check the expression of the nucleic acid molecule of interest in, for example, CHO cells, by recombining the nucleic acid molecule of interest into a CMV promoter vector (pDEST7, Figure 27, or pDESTl 2, Figure 32), or into a baculovirus vector (pDEST8, Figure 28, or pDESTlO, Figure 30) for expression in insect cells Both of these vectors will transcribe the coding sequence of your nucleic acid molecule of interest, and translate it from the ATG of the PCR product using the Kozak bases upstream of the Xmnl site. Option 5: If you wish to purify protein, for example to make antibodies, you can clone the nucleic acid molecule of interest into a His6 fusion vector, pDEST2 (Figure 22). Since the nucleic acid molecule of interest is cloned downstream of the TEV protease cleavage domain of pENTR7 (Figure 16), the amino acid sequence of the protein produced will be: [ attBl ] TEV protease NH2- MS YYHHHHHHGITSL YKKAGFE/V YFQ 1 GTM — COOH The attB site and the restriction sites used to make the Destination and Entry Vectors are translated into the underlined 11 amino acids (GITSLYKKAGF). Cleavage with TEV protease (arrow) leaves two amino acids, GT, on the amino end of the gene product. See Figure 55 for an example of a nucleic acid molecule of interest, the chloramphenicol acetyl transferase (CAT) gene, cloned into pEΝTR7 (Figure 16) as a blunt (amino)- 7.oI (carboxy) fragment, then cloned by recombination into the His6 fusion vector pDEST2 (Figure 22). Option 6: If the His6 fusion protein is insoluble, you may go on and try a GST fusion. The appropriate Destination vector is pDEST3 (Figure 23). Option 7: If you need to make RNA probes and prefer SP6 RNA polymerase, you can make the top strand RNA with your nucleic acid molecule of interest cloned into pSPORT+ (pDEST5 (Figures 25A, 25B)), and the bottom strand RNA with the nucleic acid molecule of interest cloned into pSPORT(-) (pDESTό (Figures 26A, 26B)). Opposing promoters for T7 RNA polymerase and SP6 RNA polymerase are also present in these clones. Option 8: It is often worthwhile to clone your nucleic acid molecule of interest into a variety of Destination Vectors in the same experiment. For example, if the number of colonies varies widely when the various recombination reactions are transformed into E. coli, this may be an indication that the nucleic acid molecule of interest is toxic in some contexts. (This problem is more clearly evident when a positive control gene is used for each Destination Vector.) Specifically, if many more colonies are obtained when the nucleic acid molecule of interest is recombined into pDΕST6 than in pDEST5, there is a good chance that leakiness of the lac promoter is causing some expression of the nucleic acid molecule of interest in pSPORT "+" (which is not harmful in pDESTό because the nucleic acid molecule of interest is in the opposite orientation). Example 16: Demonstration of a One-tube Transfer of a PCR Product (or Expression Clone) to Expression Clone via a Recombinational Cloning Reaction In the BxP recombination (Entry or Gateward) reaction described herein, a DNA segment flanked by attB 1 and attB2 sites in a plasmid conferring ampicillin resistance was transferred by recombination into an attP plasmid conferring kanamycin resistance, which resulted in a product molecule wherein the DNA segment was flanked by attL sites (attLl and attL2). This product plasmid comprises an "attL Entry Clone" molecule, because it can react with a "attR Destination Vector" molecule via the LxR (Destination) reaction, resulting in the transfer of the DNA segment to a new (ampicillin resistant) vector. In the previously described examples, it was necessary to transform the BxP reaction products into E. coli, select kanamycin resistant colonies, grow those colonies in liquid culture, and prepare miniprep DNA, before reacting this DNA with a Destination Vector in an LxR reaction. The goal of the following experiment was to eliminate the transformation and miniprep DNA steps, by adding the BxP Reaction products directly to an LxR Reaction. This is especially appropriate when the DNA segment flanked by attB sites is a PCR product instead of a plasmid, because the PCR product cannot give ampicillin-resistant colonies upon transformation, whereas attB plasmids (in general) carry an ampicillin resistance gene Thus use of a PCR product flanked by attB sites in a BxP Reaction allows one to select for the ampicillin resistance encoded by the desired attB product of a subsequent LxR Reaction Two reactions were prepared Reaction A. negative control, no attB PCR product, (8 μl) contained 50 ng pEZC7102 (attP Donor plasmid, confers kanamycin resistance) and 2 μl BxP Clonase (22 ng / μl Int protein and 8 ng/μl IHF protein) in BxP buffer (25 mM Tris HCl, pH 7 8, 70 mM KCl, 5 mM spermidine, 0 5 mM EDTA, 250 μg / ml BSA) Reaction B (24 μl) contained 150 ng pEZC7102, 6 μl BxP Clonase, and 120 ng of the attB -tet-PCR product in the same buffer as reaction A The attB - tet - PCR product comprised the tetracycline resistance gene of plasmid pBR322, amplified with two primers containing either attBl or attB2 sites, and having 4 Gs at their 5' ends, as described earlier The two reactions were incubated at 25°C for 30 minutes Then aliquots of these reactions were added to new components that comprised LxR Reactions or appropriate controls for the LxR Reaction Five new reactions were thus produced Reaction 1 : 5 μl of reaction A was added to a 5 μl LxR Reaction containing 25 ng Ncol-cut pEZC8402 (the attR Destination Vector plasmid) in LxR buffer (37 5 mM Tris HCl, pH 7 7, 16 5 mM ΝaCl, 35 mM KCl, 5 mM spermidine, 375 μg / ml BSA), and 1 μl of GATEWAY™ LR Clonase™ Enzyme Mix (total volume of 10 μl) Reaction 2: Same as reaction 1, except 5 μl of reaction B (positive) were added instead of reaction A (negative) Reaction 3: Same as reaction 2, except that the amounts of Νco-cut pEZC8402 and GATEWAY™ LR Clonase™ Enzyme Mix were doubled, to 50 ng and 2 μl, respectively Reaction 4: Same as reaction 2, except that 25 ng of pEZ11104 (a positive control attL Entry Clone plasmid) were added in addition to the aliquot of reaction B Reaction 5: Positive control LxR Reaction, containing 25 ng Ncol-cut pEZC8402, 25 ng pEZ11104, 37 5 mM Tris HCl pH 7 7, 16 5 mM ΝaCl, 35 mM KCl, 5 mM spermidine, 375 μg / ml BSA and 1 μl GATEWAY™ LR Clonase™ Enzyme Mix in a total volume of 5 μl All five reactions were incubated at 25 °C for 30 minutes Then, 1 μl aliquots of each of the above five reactions, plus 1 μl from the remaining volume of Reaction B, the standard BxP Reaction, were used to transform 50 μl competent DH5α E. cob DΝA and cells were incubated on ice for 15 min , heat shocked at 42 °C for 45 sec , and 450 μl SOC were added Each tube was incubated with shaking at 37° C for 60 min Aliquots of 100 μl and 400 μl of each transformation were plated on LB plates containing either 50 μg/ml kanamycin or 100 μg/ml ampicillin (see Table 2) A transformation with 10 pg of pUC19 DΝA (plated on LB-amp 100 ) served as a control on the transformation efficiency of the DH5α cells Following incubation overnight at 37°C, the number of colonies on each plate was determined Results of these reactions are shown in Table 2 Table 2* ♦ (Transformation with pUC 19 DNA yielded 1 4 x 10 9 CFU/μg DNA ) 34 of the 43 colonies obtained from Reaction 3 were picked into 2 ml Terrific Broth with 100 μg/ml ampicillin and these cultures were grown overnight, with shaking, at 37°C 27 of the 34 cultures gave at least moderate growth, and of these 24 were used to prepare miniprep DNN using the standard protocol These 24 DΝAs were initially analyzed as supercoiled (SC) DΝA on a 1 % agarose gel to identify those with inserts and to estimate the sizes of the inserts Fifteen of the 24 samples displayed SC DΝA of the size predicted (5553 bp) if tetx7102 had correctly recombined with pEZC8402 to yield tetx8402 One of these samples contained two plasmids, one of -5500 bp and a one of -3500 bp The majority of the remaining clones were approximately 4100 bp in size All 15 of the clones displaying SC DΝA of predicted size (-5500 bp) were analyzed by two different double digests with restriction endonucleases to confirm the structure of the expected product tetx8402. (See plasmid maps, Figures 57-59) In one set of digests, the DΝAs were treated with Not I and Eco Rl, which should cut the predicted product just outside both attB sites, releasing the tef insert on a fragment of 1475 bp In the second set of digests, the DNAs were digested with Notl and with Nrwl Nrul cleaves asymmetrically within the subcloned tef insert, and together with Notl will release a fragment of 1019 bp Of the 15 clones analyzed by double restriction digestion, 14 revealed the predicted sizes of fragments for the expected product Interpretation: The DΝA components of Reaction B, pEZC7102 and attB-tet-PCR, are shown in Figure 56 The desired product of BxP Reaction B is tetx7102, depicted in Figure 57 The LxR Reaction recombines the product of the BxP Reaction, tetx7102 (Figure 57), with the Destination Vector, pEZC8402, shown in Figure 58 The LxR Reaction with tetx7102 plus pEZC8402 is predicted to yield the desired product tetx8402, shown in Figure 59 Reaction 2, which combined the BxP Reaction and LxR Reaction, gave few colonies beyond those of the negative control Reaction In contrast, Reaction 3, with twice the amount of pEZC8402 (Figure 58) and LxR Clonase, yielded a larger number of colonies These colonies were analyzed further, by restriction digestion, to confirm the presence of expected product Reaction 4 included a known amount of attL Entry Clone plasmid in the combined BxP-plus-LxR reaction But reaction 4 yielded only about 1% of the colonies obtained when the same DNA was used in a LxR reaction alone, Reaction 6 This result suggests that the LxR reaction may be inhibited by components of the BxP reaction Restriction endonuclease analysis of the products of Reaction 3 revealed that a sizeable proportion of the colonies (14 of the 34 analyzed) contained the desired tef subclone, tetx8402 (Figure 59) The above results establish the feasibility of performing first a BxP recombination reaction followed by a LxR recombination reaction — in the same tube — simply by adding the appropriate buffer mix, recombination proteins, and DNAs to a completed BxP reaction This method should prove useful as a faster method to convert attB-containing PCR products into different Expression Clones, eliminating the need to isolate first the intermediate attL-PCR insert subclones, before recombining these with Destination Vectors This may prove especially valuable for automated applications of these reactions This same one-tube approach allows for the rapid transfer of nucleic acid molecules contained in attB plasmid clones into new functional vectors as well As in the above examples, attL subclones generated in a BxP Reaction can be recombined directly with various Destination Vectors in a LxR reaction The only additional requirement for using attB plasmids, instead of attB-containing PCR products, is that the Destination Vector(s) employed must contain a different selection marker from the one present on the attB plasmid itself and the attP vector Two alternative protocols for a one-tube reaction have also proven useful and somewhat more optimal than the conditions described above Alternative 1 Reaction buffer contained 50 mM Tris-HCl (pH 7 5), 50 mM NaCl, 0 25 mMEDTA, 2 5 mM spermidine, and 200 μg/ml BSA After a 16 (or 3) hour incubation of the PCR product (100 ng) + attP Donor plasmid (100 ng) + GATEWAY™ BP Clonase™ Enzyme Mix + Destination Vector (100 ng), 2 μl of GATEWAY™ LR Clonase™ Enzyme Mix (per 10 μl reaction mix) was added and the mixture was incubated an additional 6 (or 2) hours at 25 ° C Stop solution was then added as above and the mixture was incubated at 37 °C as above and transformed by electroporation with 1 μl directly into electrocompetent host cells Results of this series of experiments demonstrated that longer incubation times ( 16 hours vs 3 hours for the BP Reaction, 6 hours vs 2 hours for the LR Reaction) resulted in about twice as many colonies being obtained as for the shorter incubation times With two independent genes, 10/10 colonies having the correct cloning patterns were obtained Alternative 2 A standard BP Reaction under the reaction conditions described above for Alternative 1 was performed for 2 hours at 25 ° C Following the BP Reaction, the following components were added to the reaction mixture in a total volume of 7 μl 20 mM Tris-HCl, pH 7 5 100 mM NaCl 5 μg/ml Xis-His6 15% glycerol -1000 ng of Destination Vector The reaction mixture was then incubated for 2 hours at 25 °C, and 2 5 μl of stop solution (containing 2 μg/ml proteinase K) was added and the mixture was incubated at 37°C for an additional 10 minutes Chemically competent host cells were then transformed with 2 μl of the reaction mixture, or electrocompetent host cells (e.g. , EMax DH10B cells, Life Technologies, Inc ) were electroporated with 2 μl of the reaction mixture per 25-40 μl of cells Following transformation, mixtures were diluted with SOC, incubated at 37°C, and plated as described above on media selecting for the selection markers on the Destination Vector and the Entry clone (B x P reaction product) Analogous results to those described for Alternative 1 were obtained with these reaction conditions ~ a higher level of colonies containing correctly recombined reaction products were observed Example 17: Demonstration of a One-tube Transfer of a PCR Product (or Expression Clone) to Expression Clone via a Recombinational Cloning Reaction Single-tube transfer of PCR product DNA or Expression Clones into Expression Clones by recombinational cloning has also been accomplished using a procedure modified from that described in Example 16 This procedure is as follows •Perform a standard BP (Gateward) Reaction (see Examples 9 and 10) in 20 μl volume at 25 °C for 1 hour •After the incubation is over, take a 10 μl aliquot from the 20 μl total volume and add 1 μl of Proteinase K (2 mg/ml) and incubate at 37°C for 10 minutes This first aliquot can be used for transformation and gel assay of BP reaction analysis Plate BP reaction transformation on LB plates with Kanamycin (50 ug/ml) •Add the following reagents to the remaining 10 μl aliquot of the BP reaction 1 μl of 0 75 M NaCl 2 μl of destination vector (150 ng/μl) 4 μl of LR Clonase™ (after thawing and brief mixing) •Mix all reagents well and incubate at 25 °C for 3 hours. Stop the reaction at the end of incubation with 1 7 μl of Proteinase K (2 mg/ml) and incubate at 37°C for 10 minutes •Transform 2 μl of the completed reaction into 100 μl of competent cells Plate 100 μl and 400 μl on LB plates with Ampicilin (100 μg/ml) Notes: •If your competent cells are less than 10 8 CFU/μg, and you are concerned about getting enough colonies, you can improve the yield several fold by incubating the BP reaction for 6-20 hours Electroporation also can yield better colony output than chemical transformation •PCR products greater than about 5-6 kb show significantly lower cloning efficiency in the BP reaction In this case, we recommend using longer incubation times for both BP and LR steps •If you want to move your insert gene into several destination vectors simultaneously, then scale up the initial BP reaction volume so that you have a 10 μl aliquot for adding each destination vector Example 18: Optimization ofGA TEWA F™ Clonase™ Enzyme Compositions The enzyme compositions containing Int and IHF (for BP Reactions) were optimized using a standard functional recombinational cloning reaction (a BP reaction) between attB-containing plasmids and attP-containing plasmids, according to the following protocol Materials and Methods Substrates AttP - supercoiled pDONR201 AttB - linear - 1Kb [ 3 H]PCR product amplified from pEZC7501 Proteins IntH6 — His 6 -carboxy- tagged λ Integrase IHF ~ Integration Host Factor Clonase 50 ng/μl IntH6 and 20 ng/μl IHF, admixed in 25 mM Tris- HCl (pH 7 5), 22 mM NaCl, 5 mM EDTA, 1 mg/ml BSA, 5 mM Spermidine, and 50% glycerol Reaction Mixture (total volume of 40 μl) 1000 ng AttP plasmid 600 ng AttB [Η] PCR product 8 μl Clonase (400 ng IntH6, 160 ng IHF) in 25 mM Tris-HCl (pH 7 5), 22 mM NaCl, 5 mM EDTN 1 mg/ml BSA, 5 mM Spermidine, 5 mM DTT Reaction mixture was incubated for 1 hour at 25 °C, 4 μl of 2 μg/μl proteinase K was added and mixture was incubated for an additional 20 minutes at 37 ° C Mixture was then extracted with an equal volume of Phenol/Chloroform Isoamyl alcohol The aqueous layer was then collected, and 0 1 volumes of 3 M sodium acetate and 2 volumes of cold 100% ethanol were added Tubes were then spun in a microcentrifuge at maximum RPM for 10 minutes at room temperature Ethanol was decanted, and pellets were rinsed with 70%> ethanol and re-centrifuged as above Ethanol was decanted, and pellets were allowed to air dry for 5-10 minutes and then dissolved in 20 μl of 33 mM Tris- Acetate (pH 7 8), 66 mM potassium acetate, 10 mM magnesium acetate, 1 mM DTT, and ImM ATP 2 units of exonuclease V (e.g. , Plasmid Safe, Epicentre, Inc , Madison, WI) was then added, and the mixture was incubated at 37° C for 30 minutes Samples were then TCA-washed by spotting 30 μl of reaction mixture onto a Whatman GF/C filter, washing filters once with 10% TCA + 1 % ΝaPPi for 10 minutes, three times with 5% TCA for 5 minutes each, and twice with ethanol for 5 minutes each Filters were then dried under a heat lamp, placed into a scintillation vial, and counted on a β liquid scintillation counter (LSC) The principle behind this assay is that, after exonuclease V digestion, only double-stranded circular DΝA survives in an acid-insoluble form All DΝA substrates and products that have free ends are digested to an acid-soluble form and are not retained on the filters Therefore, only the 3 H-labeled attB linear DΝA which ends up in circular form after both inter- and intramolecular integration is complete is resistant to digestion and is recovered as acid-insoluble product Optimal enzyme and buffer formulations in the Clonase compositions therefore are those that give the highest levels of circularized 3 H-labeled attB-containing sequences, as determined by highest cpm in the LSC Although this assay was designed for optimization of GATEWAY™ BP Clonase™ Enzyme Mix compositions (Int + IHF), the same type of assay may be performed to optimize GATEWAY™ LR Clonase™ Enzyme Mix compositions (Int + IHF + Xis), except that the reaction mixtures would comprise 1000 ng of AttR (instead of AttP) and 600 ng of AttL (instead of AttB), and 40 ng of His 6 -carboxy- tagged Xis (XisH6) in addition to the IntH6 and IHF Example 19: Testing Functionality of Entry and Destination Vectors As part of assessment of the functionality of particular vectors of the invention, it is important to functionally test the ability of the vectors to recombine This assessment can be carried out by performing a recombinational cloning reaction (as schematized in Figures 2, 4, and 5A and 5B, and as described herein and in commonly owned U S Application Nos 08/486,139, filed June 1, 1995, 08/663,002, filed June 7, 1996 (now U S Patent No 5,888,732), 09/005,476, filed January 12, 1998, and 09/177,387, filed October 23, 1998, the disclosures of all of which are incorporated by reference herein in their entireties), by transforming E coli and scoring colony forming units However, an alternative assay may also be performed to allow faster, more simple assessment of the functionality of a given Entry or Destination Vector by agarose gel electrophoresis The following is a description of such an in vitro assay Materials and Methods Plasmid templates pEZC1301 (Figure 84) and pEZC1313 (Figure 85), each containing a single wild type att site, were used for the generation of PCR products containing attL or attR sites, respectively Plasmid templates were linearized with AlwNl, phenol extracted, ethanol precipitated and dissolved in TE to a concentration of 1 ng/μl PCR primers (capital letters represent base changes from wildtype): attLl gggg agcct gcttttttGtacAaa gttggcatta taaaaaagca ttgc attL2 gggg agcct gctttCttGtacAaa gttggcatta taaaaaagca ttgc attL right tgttgccggg aagctagagt aa attRl gggg Acaag ttTgtaCaaaaaagc tgaacgaga aacgtaaaat attR2 gggg Acaag ttTgtaCaaGaaagc tgaacgaga aacgtaaaat attR right ca gacggcatga tgaacctgaa PCR primers were dissolved in TE to a concentration of 500 pmol/μl. Primer mixes were prepared, consisting of attLl + attLright primers, attL2 + attLright primers, attRl + attRright primers, and attR2 + attRright primers, each mix containing 20 pmol/μl of each primer. PCR reactions: 1 μl plasmid template (1 ng) 1 μl primer pairs (20 pmoles of each) 3 μl of H 2 0 45 μl of Platinum PCR SuperMix® (Life Technologies, Inc.) Cycling conditions (performed in MJ thermocycler): 95 °C/2 minutes 94°C/30 seconds 25 cycles of 58°C/30 seconds and 72°C/1.5 minutes 72°C/5 minutes 5°C/hold The resulting attL PCR product was 1.5 kb, and the resulting attR PCR product was 1.0 kb. PCR reactions were PEG/MgCl 2 precipitated by adding 150 μl H 2 O and 100 μl of 3x PEG/ MgCl 2 solution followed by centrifugation. The PCR products were dissolved in 50 μl of TE. Quantification of the PCR product was performed by gel electrophoresis of 1 μl and was estimated to be 50-100 ng/μl. Recombination reactions of PCR products containing attL or attR sites with GATEWAY™ plasmids was performed as follows 8 μl of H 2 0 2 μl of attL or attR PCR product (100-200 ng) 2 μl of GATEWAY™ plasmid ( 100 ng) 4 μl of 5x Destination buffer 4 μl of GATEWAY™ LR Clonase™ Enzyme Mix 20 μl total volume (the reactions can be scaled down to a 5 μl total volume by adjusting the volumes of the components to about l/ of those shown above, while keeping the stoichiometries the same) Clonase reactions were incubated at 25 °C for 2 hours 2 μl of proteinase K (2 mg/ml) was added to stop the reaction 10 μl was then run on a 1 % agarose gel Positive control reactions were performed by reacting attLl PCR product (1 0 kb) with attRl PCR product (1 5 kb) and by similarly reacting attL2 PCR product with attR2 PCR product to observe the formation of a larger (2 5 kb) recombination product Negative controls were similarly performed by reacting attLl PCR product with attR2 PCR product and vice versa or reactions of attL PCR product with an attL plasmid, etc In alternative assays, to test attB Entry vectors, plasmids containing single attP sites were used Plasmids containing single att sites could also be used as recombination substrates in general to test all Entry and Destination vectors (i.e., those containing attL, attR , attB and attP sites) This would eliminate the need to do PCR reactions Results Destination and Entry plasmids when reacted with appropriate att-containing PCR products formed linear recombinant molecules that could be easily visualized on an agarose gel when compared to control reactions containing no attL or attR PCR product Thus, the functionality of Destination and Entry vectors constructed according to the invention may be determined either by carrying out the Destination or Entry recombination reactions as depicted in Figures 2, 4, and 5A and 5B, or more rapidly by carrying out the linearization assay described in this Example Example 20: PCR Cloning Using Universal Adapter-Primers As described herein, the cloning of PCR products using the GATEWAY™ PCR Cloning System (Life Technologies, Inc , Rockville, MD) requires the addition of attB sites (attBl and attB2) to the ends of gene-specific primers used in the PCR reaction The protocols described in the preceding Examples suggest that the user add 29 bp (25 bp containing the attB site plus four G residues) to the gene-specific primer It would be advantageous to high volume users of the GATEWAY™ PCR Cloning System to generate attB-containing PCR product using universal attB adapter-primers in combination with shorter gene-specific primers containing a specified overlap to the adapters The following experiments demonstrate the utility of this strategy using universal attB adapter-primers and gene-specific primers containing overlaps of various lengths from 6 bp to 18 bp The results demonstrate that gene-specific primers with overlaps of 10 bp to 18 bp can be used successfully in PCR amplifications with universal attB adapter- primers to generate full-length PCR products These PCR products can then be successfully cloned with high fidelity in a specified orientation using the GATEWAY™ PCR Cloning System Methods and Results To demonstrate that universal attB adapter-primers can be used with gene- specific primers containing partial attB sites in PCR reactions to generate full- length PCR product, a small 256 bp region of the human hemoglobin cDNA was chosen as a target so that intermediate sized products could be distinguished from full-length products by agarose gel electrophoresis The following oligonucleotides were used Bl-Hgb: GGGG ACA AGT TTG TAC AAA AAA GCA GGC T-5' -Hgb* B2-Hgb:GGGG ACC ACT TTG TAC AAG AAA GCT GGG T-3 ' -Hgb** 18B1-Hgb TG TAC AAA AAA GCA GGC T-5 ' -Hgb 18B2-Hgb TG TAC AAG AAA GCT GGG T-3 ' -Hgb 15B1-Hgb AC AAA AAA GCA GGC T-5' -Hgb 15B2-Hgb AC AAG AAA GCT GGG T-3' -Hgb 12B1-Hgb AA AAA GCA GGC T-5' -Hgb 12B2-Hgb AG AAA GCT GGG T-3 ' -Hgb HBl-Hgb A AAA GCA GGC T-5' -Hgb 11B2-Hgb G AAA GCT GGG T-3 ' -Hgb lOBl-Hgb AAA GCA GGC T-5 '-Hgb 10B2-Hgb AAA GCT GGG T-3 ' -Hgb 9B1-Hgb AA GCA GGC T-5' -Hgb 9B2-Hgb AA GCT GGG T-3 ' -Hgb 8B1-Hgb A GCA GGC T-5' -Hgb 8B2-Hgb A GCT GGG T-3 ' -Hgb 7B1-Hgb GCA GGC T-5' -Hgb 7B2-Hgb GCT GGG T-3 ' -Hgb 6B1-Hgb CA GGC T-5' -Hgb 6B2-Hgb CT GGG T-3 ' -Hgb attBl adapter: GGGG ACA AGT TTG TAC AAA AAA GCA GGC T attB2 adapter: GGGG ACC ACT TTG TAC AAG AAA GCT GGG T * ■ 5 '-Hgb = GTC ACT AGC CTG TGG AGC AAG A ** -3 '-Hgb = AGG ATG GCA GAG GGA GAC GAC A The aim of these experiments was to develop a simple and efficient universal adapter PCR method to generate attB containing PCR products suitable for use in the GATEWAY™ PCR Cloning System. The reaction mixtures and thermocycling conditions should be simple and efficient so that the universal adapter PCR method could be routinely applicable to any PCR product cloning application. PCR reaction conditions were initially found that could successfully amplify predominately full-length PCR product using gene-specific primers containing 18bp and 15 bp overlap with universal attB primers. These conditions are outlined below: 10 pmoles of gene-specific primers 10 pmoles of universal attB adapter-primers 1 ng of plasmid containing the human hemoglobin cDNA. 100 ng of human leukocyte cDNA library DNA. 5 μl of 1 Ox PLATINUM Taq HiFi® reaction buffer (Life Technologies, Inc.) 2 μl of 50 mM MgSO 4 1 μl of lO mM dNTPs 0.2 μl of PLATINUM Taq HiFi® (1.0 unit) H 2 O to 50 μl total reaction volume Cycling conditions: 95°C/5 min 94°C/15 sec 25 x 50°C/30 sec 68°C/1 min 68°C/5 min 5°C/hold To assess the efficiency of the method, 2 μl (1/25) of the 50 μl PCR reaction was electrophoresed in a 3 % Agarose- 1000 gel. With overlaps of 12 bp or less, smaller intermediate products containing one or no universal attB adapter predominated the reactions. Further optimization of PCR reaction conditions was obtained by titrating the amounts of gene-specific primers and universal attB adapter-primers. The PCR reactions were set up as outlined above except that the amounts of primers added were: 0, 1, 3 or 10 pmoles of gene-specific primers 0, 10, 30 or 100 pmoles of adapter-primers Cycling conditions 95°C/3 min 94°C/15 sec 25 x 50°C/45 sec 68°C/1 min 68°C/5 min 5°C/hold The use of limiting amounts of gene-specific primers (3 pmoles) and excess adapter-primers (30 pmoles) reduced the amounts of smaller intermediate products Using these reaction conditions the overlap necessary to obtain predominately full-length PCR product was reduced to 12 bp The amounts of gene-specific and adapter-primers was further optimized in the following PCR reactions 0, 1, 2 or 3 pmoles of gene-specific primers 0, 30, 40 or 50 pmoles of adapter-primers Cycling conditions 95°C/3 min 94°C/15 sec 25 x 48°C/1 min 68°C/1 min 68°C/5 min 5°C/hold The use of 2 pmoles of gene-specific primers and 40 pmoles of adapter- primers further reduced the amounts of intermediate products and generated predominately full-length PCR products with gene-specific primers containing an 1 1 bp overlap. The success of the PCR reactions can be assessed in any PCR application by performing a no adapter control The use of limiting amounts of gene-specific primers should give faint or barely visible bands when 1/25 to 1/10 of the PCR reaction is electrophoresed on a standard agarose gel. Addition of the universal attB adapter-primers should generate a robust PCR reaction with a much higher overall yield of product PCR products from reactions using the 18 bp, 15 bp, 12 bp, 1 1 bp and 10 bp overlap gene-specific primers were purified using the CONCERT® Rapid PCR Purification System (PCR products greater than 500 bp can be PEG precipitated) The purified PCR products were subsequently cloned into an attP containing plasmid vector using the GATEWAY™ PCR Cloning System (Life Technologies, Inc , Rockville, MD) and transformed into E. coh Colonies were selected and counted on the appropriate antibiotic media and screened by PCR for correct inserts and orientation Raw PCR products (unpurified) from the attB adapter PCR of a plasmid clone of part of the human beta-globin (Hgb) gene were also used in GATEWAY™ PCR Cloning System reactions PCR products generated with the full attB Bl/B2-Hgb, the 12B1/B2, 11B1/B2 and 10B1/B2 attB overlap Hgb primers were successfully cloned into the GATEWAY™ pENTR21 attP vector (Figure 49) 24 colonies from each (24 x 4 = 96 total) were tested and each was verified by PCR to contain correct inserts The cloning efficiency expressed as cfu/ml is shown below Interestingly, the overlap PCR products cloned with higher efficiency than did the full attB PCR product Presumably, and as verified by visualization on agarose gel, the adapter PCR products were slightly cleaner than was the full attB PCR product The differences in colony output may also reflect the proportion of PCR product molecules with intact attB sites Using the attB adapter PCR method, PCR primers with 12 bp attB overlaps were used to amplify cDNAs of different sizes (ranging from 1 to 4 kb) from a leukocyte cDNA library and from first strand cDNA prepared from HeLa total RNA While three of the four cDNAs were able to be amplified by this method, a non-specific amplification product was also observed that under some conditions would interfere with the gene-specific amplification This non-specific product was amplified in reactions containing the attB adapter-primers alone without any gene-specific overlap primers present The non-specific amplification product was reduced by increasing the stringency of the PCR reaction and lowering the attB adapter PCR primer concentration These results indicate that the adapter-primer PCR approach described in this Example will work well for cloned genes These results also demonstrate the development of a simple and efficient method to amplify PCR products that are compatible with the GATEWAY™ PCR Cloning System that allows the use of shorter gene-specific primers that partially overlap universal attB adapter-primers In routine PCR cloning applications, the use of 12 bp overlaps is recommended The methods described in this Example can thus reduce the length of gene-specific primers by up to 17 residues or more, resulting in a significant savings in oligonucleotide costs for high volume users of the GATEWAY™ PCR Cloning System In addition, using the methods and assays described in this Example, one of ordinary skill can, using only routine experimentation, design and use analogous primer-adapters based on or containing other recombination sites or fragments thereof, such as αttL, ttR, αttP, lox, FRT, etc Example 21: Mutational Analysis of the Bacteriophage Lambda attL and attR Sites: Determinants of att Site Specificity in Site-specific Recombination To investigate the determinants of αtt site specificity, the bacteriophage lambda αttL and αttR sites were systematically mutagenized As noted herein, the determinants of specificity have previously been localized to the 7 bp overlap region (TTT AT AC, which is defined by the cut sites for the integrase protein and is the region where strand exchange takes place) within the 15 bp core region (GCTTTTTTATACTAA) which is identical in all four lambda att sites, αttB, αttP, αttL and αttR This core region, however, has not heretofore been systematically mutagenized and examined to define precisely which mutations produce unique changes in att site specificity Therefore, to examine the effect of αtt sequence on site specificity, mutant αttL and αttR sites were generated by PCR and tested in an in vitro site-specific recombination assay In this way all possible single base pair changes within the 7 bp overlap region of the core att site were generated as well as five additional changes outside the 7 bp overlap but within the 15 bp core att site Each αttL PCR substrate was tested in the in vitro recombination assay with each of the αttR PCR substrates Methods To examine both the efficiency and specificity of recombination of mutant αttL and αttR sites, a simple in vitro site-specific recombination assay was developed Since the core regions of αttL and αttR lie near the ends of these sites, it was possible to incorporate the desired nucleotide base changes within PCR primers and generate a series of PCR products containing mutant αttL and αttR sites PCR products containing αttL and αttR sites were used as substrates in an in vitro reaction with GATEWAY™ LR Clonase™ Enzyme Mix (Life Technologies, Inc , Rockville, MD) Recombination between a 1 5 kb αttL PCR product and a 1 0 kb αttR PCR product resulted in a 2 5 kb recombinant molecule that was monitored using agarose gel electrophoresis and ethidium bromide staining Plasmid templates pEZC1301 (Figure 84) and pEZC1313 (Figure 85), each containing a single wild type αttL or αttR site, respectively, were used for the generation of recombination substrates The following list shows primers that were used in PCR reactions to generate the αttL PCR products that were used as substrates in L x R Clonase reactions (capital letters represent changes from the wild-type sequence, and the underline represents the 7 bp overlap region within the 15 bp core att site, a similar set of PCR primers was used to prepare the αttR PCR products containing matching mutations) GATEWAY™ sites (note: attL2 sequence in GATEWAY™ plasmids begins "accca" while the attL2 site in this example begins "agccf'to reflect wild-type attL outside the core region.) : attLl: gggg agcct cttttttGtacAaa gttggcatta taaaaaagca ttgc attL2: gggg agcct gctttCttGtacAaa gttggcatta taaaaa- agca ttgc Wild-type: attLO : gggg agcct gcttttttatactaa gttggcatta taaaaaagca ttgc Single base changes from wild-type : attLTIA: gggg agcct gctttAttatactaa gttggcatta taaaaaagca ttgc attLTIC: gggg agcct gctttCttatactaa gttggcatta taaaaaagca ttgc attLTIG: gggg agcct gctttGttatactaa gttggcatta taaaaaagca ttgc attLT2A: gggg agcct crcttttAtatactaa gttggcatta taaaaaagca ttgc attLT2C: gggg agcct gcttttCtatactaa gttggcatta taaaaa- agca ttgc attLT2G: gggg agcct gcttttGtatactaa gttggcatta taaaaaagca ttgc attLT3A: gggg agcct gctttttAatactaa gttggcatta taaaaaagca ttgc attLT3C: gggg agcct crctttttCatactaa gttggcatta taaaaaagca ttgc attLT3G: gggg agcct gctttttGatactaa gttggcatta taaaaaagca ttgc attLA4C: gggg agcct gcttttttCtactaa gttggcatta taaaaaagca ttgc attLA4G: gggg agcct gcttttttGtactaa gttggcatta taaaaaagca ttgc attLA4T: gggg agcct gcttttttTtactaa gttggcatta taaaa- aagca ttgc attLT5A: gggg agcct crcttttttaAactaa gttggcatta taaaaaagca ttgc attLT5C: gggg agcct gcttttttaCactaa gttggcatta taaaaaagca ttgc attLT5G: gggg agcct gcttttttaGactaa gttggcatta taaaaaagca ttgc attLA6C: gggg agcct gcttttttatCctaa gttggcatta taaaa- aagca ttgc attLA6G: gggg agcct gcttttttatGctaa gttggcatta taaaaaagca ttgc attLA6T: gggg agcct gcttttttatTctaa gttggcatta taaaaaagca ttgc attLC7A: gggg agcct gcttttttataAtaa gttggcatta taaaa- aagca ttgc atϋLC7G: gggg agcct gcttttttataGtaa gttggcatta taaaaaagca ttgc attLC7T: gggg agcct gcttttttataTtaa gttggcatta taaaaaagca ttgc Single base changes outside of the 7 bp overlap: attLδ: gg g agcct Acttttttatactaa gttggcatta taaaaaagca ttgc attL9: gggg agcct gcCtttttatactaa gttggcatta taaaaa- agca ttgc attLlO: gggg agcct gcttCtttatactaa gttggcatta taaaaaagca ttgc attL14: gggg agcct gcttttttatacCaa gttggcatta taaaaaagca ttgc attLl5: gggg agcct gcttttttatactaG gttggcatta taaaaa- agca ttgc Note additional vectors wherein the first nine bases are gggg agcca (i.e. , substituting an adenine for the thymine in the position immediately preceding the 15-bp core region), which may or may not contain the single base pair substitutions (or deletions) outlined above, can also be used in these experiments Recombination reactions of αttL- and αttR-containing PCR products was performed as follows 8 μl of H 2 0 2 μl of attL PCR product (100 ng) 2 μl of attR PCR product (100 ng) 4 μl of 5x buffer 4 ul of GATEWAY™ LR Clonase™ Enzyme Mix 20 μl total volume Clonase reactions were incubated at 25°C for 2 hours 2 μl of 1 OX Clonase stop solution (proteinase K, 2 mg/ml) were added to stop the reaction 10 μl were run on a 1 % agarose gel Results Each αttL PCR substrate was tested in the in vitro recombination assay with each of the αttR PCR substrates Changes within the first three positions of the 7 bp overlap (TTT AT AC) strongly altered the specificity of recombination These mutant att sites each recombined as well as the wild-type, but only with their cognate partner mutant, they did not recombine detectably with any other att site mutant In contrast, changes in the last four positions (TTTATAC) only partially altered specificity, these mutants recombined with their cognate mutant as well as wild-type att sites and recombined partially with all other mutant att sites except for those having mutations in the first three positions of the 7 bp overlap Changes outside of the 7 bp overlap were found not to affect specificity of recombination, but some did influence the efficiency of recombination Based on these results, the following rules for att site specificity were determined »Only changes within the 7 bp overlap affect specificity •Changes within the first 3 positions strongly affect specificity •Changes within the last 4 positions weakly affect specificity Mutations that affected the overall efficiency of the recombination reaction were also assessed by this method In these experiments, a slightly increased (less than 2-fold) recombination efficiency with αttLTl A and αttLC7T substrates was observed when these substrates were reacted with their cognate αttR partners Also observed were mutations that decreased recombination efficiency (approximately 2-3 fold), including αttLA6G, αttL 14 and αttL 15 These mutations presumably reflect changes that affect Int protein binding at the core att site The results of these experiments demonstrate that changes within the first three positions of the 7 bp overlap (TTT AT AC) strongly altered the specificity of recombination (i.e., att sequences with one or more mutations in the first three thymidines would only recombine with their cognate partners and would not cross-react with any other att site mutation) In contrast, mutations in the last four positions (TTTATAC) only partially altered specificity (i.e., att sequences with one or more mutations in the last four base positions would cross-react partially with the wild-type att site and all other mutant att sites, except for those having mutations in one or more of the first three positions of the 7 bp overlap) Mutations outside of the 7 bp overlap were not found to affect specificity of recombination, but some were found to influence (/. e. , to cause a decrease in) the efficiency of recombination Example 22: Discovery of Att Site Mutations That Increase the Cloning Efficiency of GATEWAY™ Cloning Reactions In experiments designed to understand the determinants of att site specificity, point mutations in the core region of αttL were made Nucleic acid molecules containing these mutated αttL sequences were then reacted in an LR reaction with nucleic acid molecules containing the cognate αttR site (/. e. , an αttR site containing a mutation corresponding to that in the αttL site), and recombinational efficiency was determined as described above Several mutations located in the core region of the att site were noted that either slightly increased (less than 2-fold) or decreased (between 2-4-fold) the efficiency of the recombination reaction (Table 3) Table 3. Effects of attL mutations on Recombination Reactions. Site Sequence Effect on Recombination attLO agcctgcttttttatactaagttggcatta attL5 agcct get ttAttatactaagttggcatta slightly increased attLδ agcctgcttttttataTtaagttggcatta slightly increased attL13 agcctgcttttttatGctaagttggcatta decreased attL14 agcctgcttttttatacCaagttggcatta decreased attL15 agcctgcttttttatactaGgttggcatta decreased consensus CAACTTnnTnnnAnnAAGTTG It was also noted that these mutations presumably reflected changes that either increased or decreased, respectively, the relative affinity of the integrase protein for binding the core att site A consensus sequence for an integrase core- binding site (CAACTTNNT) has been inferred in the literature but not directly tested (see, e.g., Ross and Landy, Cell 33.261-212 (1983)) This consensus core integrase-binding sequence was established by comparing the sequences of each of the four core att sites found in attP and attB as well as the sequences of five non-att sites that resemble the core sequence and to which integrase has been shown to bind in vitro These experiments suggest that many more att site mutations might be identified which increase the binding of integrase to the core att site and thus increase the efficiency of GATEWAY™ cloning reactions Example 23: Effects of Core Region Mutations on Recombination Efficiency To directly compare the cloning efficiency of mutations in the att site core region, single base changes were made in the attB2 site of an attBl-TET-attB2 PCR product Nucleic acid molecules containing these mutated αttB2 sequences were then reacted in a BP reaction with nucleic acid molecules containing non- cognate αttP sites (i.e., wildtype αttP2), and recombinational efficiency was determined as described above The cloning efficiency of these mutant attB2 containing PCR products compared to standard attB 1 -TET-attB2 PCR product are shown in Table 4 7αb/e 4. Efficiency of Recombination With Mutated attB 2 Sites. Cloning Site Sequence Mutation Efficiency attBO tcaagttagtataaaaaagcaggct attB 1 ggggacaagtttgtacaaaaaagcaggct attB2 ggggaccactttgtacaagaaagctgggt 100% attB2 1 ggggaAcactttgtacaagaaagctgggt C→A 40% attB 2.2 ggggacAactttgtacaagaaagctgggt C→A 131% attB2 3 ggggaccCctttgtacaagaaagctgggt A→C 4% attB2 4 ggggaccaAttt tacaagaaagctgggt C->A 11% attB2.5 ggggaccacGttgtacaagaaagctgggt T→G 4% attB2 6 ggggaccactGtgtacaagaaagctgggt T→G 6% attB2 7 ggggaccacttGgtacaagaaagctgggt T→G 1% attB2.8 ggggaccactttTtacaagaaagctgggt G→T 0.5% As noted above, a single base change in the attB2.2 site increased the cloning efficiency of the attBl-TET-attB2.2 PCR product to 131% compared to the attBl-TET-attB2 PCR product Interestingly, this mutation changes the integrase core binding site of attB2 to a sequence that matches more closely the proposed consensus sequence Additional experiments were performed to directly compare the cloning efficiency of an attB 1 -TET-attB2 PCR product with a PCR product that contained attB sites containing the proposed consensus sequence (see Example 22) of an integrase core binding site The following attB sites were used to amplify attB- TET PCR products attBl ggggacaagtttgtacaaaaaagcaggct attBl .6 ggggacaaCtttgtacaaaaaagTTggct attB2 ggggaccactttgtacaagaaagctgggt attB2.10 ggggacAactttgtacaagaaagTtgggt BP reactions were carried out between 300 ng (100 frnoles) of pDONR201 (Figure 49A) with 80 ng (80 frnoles) of attB-TET PCR product in a 20 μl volume with incubation for 1 5 hrs at 25 ° C, creating pENTR201 -TET Entry clones A comparison of the cloning efficiencies of the above-noted attB sites in BP reactions is shown in Table 5 Table 5. Cloning efficiency ofBP Reactions. These results demonstrate that attB PCR products containing sequences that perfectly match the proposed consensus sequence for integrase core binding sites can produce Entry clones with four-fold higher efficiency than standard Gateway attBl and attB2 PCR products The entry clones produced above were then transferred to pDEST20 (Figure 40A) via LR reactions (300 ng (64 frnoles) pDEST20 mixed with 50 ng (77 frnoles) of the respective pENTR201-TET Entry clone in 20 μl volume, incubated for 1 hr incubation at 25 °C) The efficiencies of cloning for these reactions are compared in Table 6 Table 6. Cloning Efficiency ofLR Reactions. These results demonstrate that the mutations introduced into attB 1 6 and attB2 10 that transfer with the gene into entry clones slightly increase the efficiency of LR reactions Thus, the present invention encompasses not only mutations in αttB sites that increase recombination efficiency, but also to the corresponding mutations that result in the αttL sites created by the BP reaction To examine the increased cloning efficiency of the attB 1 6-TET-attB2 10 PCR product over a range of PCR product amounts, experiments analogous to those described above were performed in which the amount of attB PCR product was titrated into the reaction mixture The results are shown in Table 7 Table 7. Titration of attB PCR products. These results demonstrate that as much as a six-fold increase in cloning efficiency is achieved with the attBl 6-TET-attB2 10 PCR product as compared to the standard attBl-TET-attB2 PCR product at the 20 ng amount Example 24: Determination of attB Sequence Requirements for Optimum Recombination Efficiency To examine the sequence requirements for attB and to determine which attB sites would clone with the highest efficiency from populations of degenerate attB sites, a series of experiments was performed. Degenerate PCR primers were designed which contained five bases of degeneracy in the B-arm of the attB site. These degenerate sequences would thus transfer with the gene into Entry clone in BP reactions and subsequently be transferred with the gene into expression clones in LR reactions. The populations of degenerate attB and attL sites could thus be cycled from attB to attL back and forth for any number of cycles. By altering the reaction conditions at each transfer step (for example by decreasing the reaction time and/or decreasing the concentration of DNA) the reaction can be made increasingly more stringent at each cycle and thus enrich for populations of attB and attL sites that react more efficiently. The following degernerate PCR primers were used to amplify a 500 bp fragment from pUC18 which contained the lacZ alpha fragment (only the attB portion of each primer is shown): attBl GGGG ACAAGTTTGTACAAA AAAGC AGGCT attBlnl6-20 GGGG ACAAGTTTGTACAAA nnnnn AGGCT attBln21-25 GGGG ACAAGTTTGTACAAA AAAGC nnnnn attB2 GGGG ACCACTTTGTACAAG AAAGC TGGGT attB2nlδ-20 GGGG ACCACTTTGTACAAG nnnnn TGGGT attB2n21-25 GGGG ACCACTTTGTACAAG AAAGC nnnnn The starting population size of degenerate att sites is 4 5 or 1024 molecules. Four different populations were transferred through two BP reactions and two LR reactions. Following transformation of each reaction, the population of transformants was amplified by growth in liquid media containing the appropriate selection antibiotic. DNA was prepared from the population of clones by alkaline lysis miniprep and used in the next reaction. The results of the BP and LR cloning reactions are shown below. BP-1, overnight reactions These results demonstrate that at each successive transfer, the cloning efficiency of the entire population of att sites increases, and that there is a great deal of flexibility in the definition of an αttB site. Specific clones may be isolated from the above reactions, tested individually for recombination efficiency, and sequenced Such new specificities may then be compared to known examples to guide the design of new sequences with new recombination specificities In addition, based on the enrichment and screening protocols described herein, one of ordinary skill can easily identify and use sequences in other recombination sites, e.g. , other att sites, lox, FRT, etc , that result in increased specificity in the recombination reactions using nucleic acid molecules containing such sequences Example 25: Design of att Site PCR Adapter-Primers Additional studies were performed to design gene-specific primers with 12bp of attB l and attB2 at their 5'-ends The optimal primer design for att- containing primers is the same as for any PCR primers the gene-specific portion of the primers should ideally have a Tm of> 50°C at 50 mM salt (calculation of Tm is based on the formula 59 9 + 41(%GC) - 675/n) Primers 12bp attBl AA ANA GCA GGC TΝΝ - forward gene-specific primer 12bp attB2 A GAA AGC TGG GTΝ - reverse gene-specific primer attBl adapter primer GGGGACAAGTTTGTACAAAAAAGCAGGCT attB2 adapter primer GGGGACCACTTTGTACAAGAAAGCTGGGT Protocol (1) Mix 200 ng of cDΝA library or 1 ng of plasmid clone DΝA (alternatively, genomic DΝA or RΝA could be used) with 10 pmoles of gene specific primers in a 50 μl PCR reaction, using one or more polypeptides having DΝA polymerase activity such as those described herein (The addition of greater than 10 pmoles of gene-specific primers can decrease the yield of attB PCR product In addition, if RΝA is used, a standard reverse transcriptase-PCR (RT- PCR) protocol should be followed, see, e.g., Gerard, G F , et al, FOCUS 11 60 (1989), Myers, T W , and Gelfand, D H , Biochem. 30 7661 (1991), Freeman, W ' N , etal, BioTechnique 20 782(1996), and U S ApplicationNo 09/064,057, filed April 22, 1998, the disclosures of all of which are incorporated herein by reference ) I s ' PCR profile (a) 95 °C for 3 minutes (b) 10 cycles of (i) 94°C for 15 seconds (ii) 50°C* for 30 seconds (iii) 68 °C for 1 minute/kb of target amplicon (c) 68 °C for 5 minutes (d) 10°C hold *The optimal annealing temperature is determined by the calculated Tm of the gene-specific part of the primer (2) Transfer 10 μl to a 40 μl PCR reaction mix containing 35 pmoles each of the attB l and attB2 adapter primers 2 nd PCR profile (a) 95 °C for 1 minute (b) 5 cycles of (i) 94 °C for 15 seconds (ii) 45 °C* for 30 seconds (iii) 68 °C for 1 minute/kb of target amplicon (c) 15-20 cycles** of (i) 94°C for 15 seconds (ii) 55 °C* for 30 seconds (iii) 68 °C for 1 minute/kb of target amplicon (d) 68 °C for 5 minutes (e) 10°C hold *The optimal annealing temperature is determined by the calculated Tm of the gene-specific part of the primer **15 cycles is sufficient for low complexity targets Notes 1 It is useful to perform a no-adapter primer control to assess the yield of attB PCR product produced 2 Linearized template usually results in slightly greater yield of PCR product Example 26: One-Tube Recombinational Cloning Using the GATEWAY™ Cloning System To provide for easier and more rapid cloning using the GATEWAY™ cloning system, we have designed a protocol whereby the BP and LR reactions may be performed in a single tube (a "one-tube" protocol) The following is an example of such a one-tube protocol, in this example, an aliquot of the BP reaction is taken before adding the LR components, but the BP and LR reactions may be performed in a one-tube protocol without first taking the BP aliquot Reaction Component Volume attB DNA (100-200 ng/25 μl reaction) 1-12 5 μl attP DNA (pDONR201) 150 ng/μl 2 5 μl 5X BP Reaction Buffer 5 0 μl Tris-EDTA (to 20 μl) BP Clonase 5 0 μl Total vol 25 μl After the above components were mixed in a single tube, the reaction mixtures were incubated for 4 hours at 25 °C A 5 μl aliquot of reaction mixture was removed, and 0 5 μl of 10X stop solution was added to this reaction mixture and incubated for 10 minutes at 37°C Competent cells were then transformed with 1-2 μl of the BP reaction per 100 μl of cells, this transformation yielded colonies of Entry Clones for isolation of individual Entry Clones and for quantitation of the BP Reaction efficiency To the remaining 20 μl of BP reaction mixture, the following components of the LR reaction were added Reaction Component Final Concentration Volume Added NaCl 0 75 M l μl Destination Vector 150 ng/ul 3 μl LR Clonase 6 μl Total vol 30 μl After the above components were mixed in a single tube, the reaction mixtures were incubated for 2 hours at 25 °C 3 μl of 10X stop solution was added, and the mixture was incubated for 10 minutes at 37°C Competent cells were then transformed with 1-2 μl of the reaction mixture per 100 μl of cells Notes 1 If desired, the Destination Vector can be added to the initial BP reaction 2 The reactions can be scaled down by 2x, if desired 3 Shorter incubation times for the BP and/or LR reactions can be used (scaled to the desired cloning efficiencies of the reaction), but a lower number of colonies will typically result 4 To increase the number of colonies obtained by several fold, incubate the BP reaction for 6-20 hours and increase the LR reaction to 3 hours Electroporation also works well with 1-2 ul of the PK-treated reaction mixture 5 PCR products greater than about 5 kb may show significantly lower cloning efficiency in the BP reaction In this case, we recommend using a one-tube reaction with longer incubation times (e.g., 6-18 hours) for both the BP and LR steps Example 27: Relaxation of Destination Vectors During the LR Reaction To further optimize the LR Reaction, the composition of the LR Reaction buffer was modified from that described above and this modified buffer was used in a protocol to examine the impact of enzymatic relaxation of Destination Vectors during the LR Reaction LR Reactions were set up as usual (see, e.g. , Example 6), except that 5X BP Reaction Buffer (see Example 5) was used for the LR Reaction To accomplish Destination Vector relaxation during the LR Reaction, Topoisomerase I (Life Technologies, Inc , Rockville, MD, Catalogue No 38042- 016) was added to the reaction mixture at a final concentration of ~15U per μg of total DNA in the reaction (for example, for reaction mixtures with a total of 400ng DNA in the 20 μl LR Reaction, ~6units of Topoisomerase I was added) Reaction mixtures were set up as follows Reaction Component Volume ddH 2 O 6 5 μl 4X BP Reaction Buffer 5 μl lOOng single chain/linear pENTR CAT, 50 ng/μl 2 μl 300ng single chain/linear pDEST6, 150ng/μl 2 μl Topoisomerase I, 15 U/ml 0 5 μl LR Clonase 4 μl Reaction mixtures were incubated at 25°C for lhour, and 2 μl of 2 μg/μl Proteinase K was then added and mixtures incubated for 10 minutes at 37 °C to stop the LR Reaction Competent cells were then transformed as described in the preceding examples The results of these studies demonstrated that relaxation of substrates in the LR reaction using Topoisomerase I resulted in a 2- to 10-fold increase in colony output compared to those LR reactions performed without including Topoisomerase I. Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. 167. 1 Applicant's or agent's file International appli JMfl-.tl reference number 0942.-,o8PC03 Rfflfe 00 / 054 3 2 INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL , REC'D 1 7 APR 2000 (PCT Rule \3bts) WfPO PCT A. The indications made below relate to the microorganism referred to in the descπption on page 52 , line 31 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet E Name of depositary institution Agricultural Research Culture Collection (NRRL) International Depository Authoπty Address of depositary institution (including postal code and country) 1815 N University Street Peoπa, Illinois 61604 United States of Ameπca Date of deposit Accession Number February 27, 1999 NRRL B-30099 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet □ Escheπchia coli DB3 l(pAHPKan) or Escheπchia coli DB3 l(pAttPKan) D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications me not for alt designated States) E. SEPARATE FURNISHING OF INDICATIONS to,, blank ι fnoi applicable, The indications listed below will be submitted to the international Bureau later (specif) the general nature of the indications, e g . "Accession Number of Deposit") Form PCT/RO/13 4 (July 1998) mo-30099 Form PCT RO I A , Julv 1998) mo 30100 INDICATIONS RELATING TO DEPOSITED MICROORGANISM FormPCT/Rθ ' 134ιJ_i> 1998) mo-30101 167. 4 Applicant's or agent's file International reference number 0942.468PC03 rømjs No tL u O / 054 3 2 REC'D 1 7 APR 2000 INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL W O (PCT Rule \3bιs) PCT A. The indications made below relate to the microorganism referred to in the descπption on page 55 line 16 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet 1 Name of depositary institution Agπcultural Research Culture Collection (NRRL) Intemationai Depository Authonty Address of depositary institution (including postal code and country) 1815 N. University Street Peoπa, Illinois 61604 United States of Ameπca Date of deposit Accession Number February 27, 1999 NRRL B-30102 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet □ Escheπchia coli DB3 l(pENTR-3C) D. DESIG ATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the international Bureau later (specify the general nature of the indications, e g , "Accession Number of Deposit") For receiving Office use only For International Bureau use only S This sheet w as received with the international application O This sheet was received by the International Bureau on Authoπzed officer - - Authoπzed officer Form PCT RO 134 1 98) mo-30102 For International Bureau use only □ This sheet was received bv the Intemationai Bureau on Authoπzed officer Form PCT RO/134 1998) mo-30101 167. 6 Applicant's or agent's file , International a reference number 0942.468PC03 ?m§°±Λ0lQ 54 3 2 INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL y/ Q (PCT Rule \3bιs) A. The indications made below relate to the microorganism referred to in the descπption on page 54 9 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet G3 Name of depositary institution Agricultural Research Culture Collection (NRRL) International Depository Authoπty Address of depositary institution (including postal code and country) 1815 N Univ ersity Street Peoπa, Illinois 61604 United States of Ameπca Date of deposit Accession Number February 27, 1999 NRRL B-30104 C. ADDITIONAL INDICATIONS (leave blank f not applicable) This information is continued on an additional sheet Q Escheπchia coh DB3 l(pEZC15102) D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank , f not applicable) The indications listed below will be submitted to the international Bureau later (specify the general nature of the indications, e g "Accession Number of Deposit") For International Bureau use only □ This sheet was received by the International Bureau on Authoπzed officer Form PCT RO 13 ljh 1998) mo 0104 167. 7 Applicant's or agent's file International app reference number 0942.468PC03 « § tl 00 / 054 3 2 INDICATI A. The indications made below relate to the microorganism referred to in the descπption on page 54 line 9 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution Agncultural Research Culture Collection (NRRL) International Depository Authoπty Address of depositary institution (including postal code and country) 1815 N University Street Peona, Illinois 61604 United States of Ameπca Date of deposit Accession Number February 27, 1999 NRRL B-30105 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet □ Escheπchia coh DB3 l(pEZC15103) D. DESIG ATED STATES FOR WHICH INDICATIONS ARE MADE dfthe indications are not for all designated Stales) E. SEPARATE FURNISHING OF INDICATIONS (leave blank , f no, applicable) The indications listed below will be submitted to the international Bureau later (specify the general nature of the indications e g "Accession Number of Deposit") For receiving Office use only For International Bureau use only W This sheet w s received with the international application □ This sheet was received by the International Bureau on Authorized officer -w Authorized officer Form PCT/RO 134 (July 1998) mo-30105 Form PCT/RO/134 (July 1998) mo-30108

Claims: -168- WHAT IS CLAIMED IS: 1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group of nucleotide sequences consisting of an attB 1 nucleotide sequence as set forth in Figure 9, an attB2 nucleotide sequence as set forth in Figure 9, an attPl nucleotide sequence as set forth in Figure 9, an attP2 nucleotide sequence as set forth in Figure 9, an attLl nucleotide sequence as set forth in Figure 9, an attL2 nucleotide sequence as set forth in Figure 9, an attRl nucleotide sequence as set forth in Figure 9, an attR2 nucleotide sequence as set forth in Figure 9, a polynucleotide complementary thereto, and a mutant, fragment, or derivative thereof. 2. An isolated nucleic acid molecule comprising an attBl nucleotide sequence as set forth in Figure 9, a polynucleotide complementary thereto, or a mutant, fragment, variant or derivative thereof. 3. An isolated nucleic acid molecule comprising an attB2 nucleotide sequence as set forth in Figure 9, a polynucleotide complementary thereto, or a mutant, fragment, variant or derivative thereof. 4. An isolated nucleic acid molecule comprising an attPl nucleotide sequence as set forth in Figure 9, a polynucleotide complementary thereto, or a mutant, fragment, variant or derivative thereof. 5. An isolated nucleic acid molecule comprising an attP2 nucleotide sequence as set forth in Figure 9, a polynucleotide complementary thereto, or a mutant, fragment, variant or derivative thereof. 6. An isolated nucleic acid molecule comprising an attLl nucleotide sequence as set forth in Figure 9, a polynucleotide complementary thereto, or a mutant, fragment, variant or derivative thereof. -169- 7 An isolated nucleic acid molecule comprising an attL2 nucleotide sequence as set forth in Figure 9, a polynucleotide complementary thereto, or a mutant, fragment, variant or derivative thereof 8 An isolated nucleic acid molecule comprising an attRl nucleotide sequence as set forth in Figure 9, a polynucleotide complementary thereto, or a mutant, fragment, variant or derivative thereof 9 An isolated nucleic acid molecule comprising an attR2 nucleotide sequence as set forth in Figure 9, a polynucleotide complementary thereto, or a mutant, fragment, variant or derivative thereof 10 The isolated nucleic acid molecule of claim 1, further comprising one or more functional or structural nucleotide sequences selected from the group consisting of one or more multiple cloning sites, one or more localization signals, one or more transcription termination sites, one or more transcriptional regulatory sequences, one or more translational signals, one or more origins of replication, one or more fusion partner peptide-encoding nucleic acid molecules, one or more protease cleavage sites, and one or more 5' polynucleotide extensions 11 The nucleic acid molecule of claim 10, wherein said transcriptional regulatory sequence is a promoter, an enhancer, or a repressor 12 The nucleic acid molecule of claim 10, wherein said fusion partner peptide-encoding nucleic acid molecule encodes glutathione S-transferase (GST), hexahistidine (His 6 ), or thioredoxin (Trx) 13 The nucleic acid molecule of claim 10, wherein said 5' polynucleotide extension consists of from one to five nucleotide bases 14 The nucleic acid molecule of claim 13, wherein said 5' polynucleotide extension consists of four or five guanine nucleotide bases -170- 15 A primer nucleic acid molecule suitable for amplifying a target nucleotide sequence, comprising the isolated nucleic acid molecule of claim 1 or a portion thereof linked to a target-specific nucleotide sequence useful in amplifying said target nucleotide sequence 16 The primer nucleic acid molecule of claim 15, wherein said primer comprises an attB 1 nucleotide sequence having the sequence shown in Figure 9 or a portion thereof, or a polynucleotide complementary to the sequence shown in Figure 9 or a portion thereof 17 The primer nucleic acid molecule of claim 15, wherein said primer comprises an attB2 nucleotide sequence having the sequence shown in Figure 9 or a portion thereof, or a polynucleotide complementary to the sequence shown in Figure 9 or a portion thereof 18 The primer nucleic acid molecule of claim 15, further comprising a 5' terminal extension of four or five guanine bases 19 A vector comprising the isolated nucleic acid molecule of claim 1 20 The vector of claim 19, wherein said vector is an Expression Vector 21 A host cell comprising the isolated nucleic acid molecule of claim 1 or the vector of claim 19 22 A method of synthesizing or amplifying one or more nucleic acid molecules comprising (a) mixing one or more nucleic acid templates with at least one polypeptide having polymerase or reverse transcriptase activity and at least a first primer comprising a template-specific sequence that is complementary to or capable of hybridizing to said -171- templates and at least a second primer comprising all or a portion of a recombination site wherein said at least a portion of said second primer is homologous to or complementary to at least a portion of said first primer; and (b) incubating said mixture under conditions sufficient to synthesize or amplify one or more nucleic acid molecules complementary to all or a portion of said templates and comprising one or more recombination sites or portions thereof at one or both termini of said molecules. 23. A method of synthesizing or amplifying one or more nucleic acid molecules comprising: (a) mixing one or more nucleic acid templates with at least one polypeptide having polymerase or reverse transcriptase activity and at least a first primer comprising a template-specific sequence that is complementary to or capable of hybridizing to said templates and at least a portion of a recombination site, and at least a second primer comprising all or a portion of a recombination site wherein said at least a portion of said recombination site on said second primer is complementary to or homologous to at least a portion of said recombination site on said first primer; and (b) incubating said mixture under conditions sufficient to synthesize or amplify one or more nucleic acid molecules complementary to all or a portion of said templates and comprising one or more recombination sites or portions thereof at one or both termini of said molecules. 24. A method of amplifying or synthesizing one or more nucleic acid molecules comprising: (a) mixing one or more nucleic acid templates with at least one polypeptide having polymerase or reverse transcriptase activity -172- and one or more first primers comprising at least a portion of a recombination site and a template-specific sequence that is complementary to or capable of hybridizing to said template, (b) incubating said mixture under conditions sufficient to synthesize or amplify one or more first nucleic acid molecules complementary to all or a portion of said templates wherein said molecules comprise at least a portion of a recombination site at one or both termini of said molecules, (c) mixing said molecules with one or more second primers comprising one or more recombination sites, wherein said recombination sites of said second primers are homologous to or complementary to at least a portion of said recombination sites on said first nucleic acid molecules, and (d) incubating said mixture under conditions sufficient to synthesize or amplify one or more second nucleic acid molecules complementary to all or a portion of said first nucleic acid molecules and which comprise one or more recombination sites at one or both termini of said molecules 25 A polypeptide encoded by the isolated nucleic acid molecule of any one of claims 1-10 26 An isolated nucleic acid molecule comprising one or more att recombination sites comprising at least one mutation in its core region that increases the specificity of interaction between said recombination site and a second att recombination site 27 The isolated nucleic acid molecule of claim 26, wherein said mutation is at least one substitution mutation of at least one nucleotide in the seven basepair overlap region of said core region of said recombination site -173- 28 The isolated nucleic acid molecule of claim 26, wherein said nucleic acid molecule comprises the sequence NNNATAC, wherein "N" refers to any nucleotide with the proviso that if one of the first three nucleotides in the consensus sequence is a T/U, then at least one of the other two of the first three nucelotides is not a T U 29 An isolated nucleic acid molecule comprising one or more mutated att recombination sites comprising at least one mutation in its core region that enhances the efficiency of recombination between a first nucleic acid molecule comprising said mutated att recombination site and a second nucleic acid molecule comprising a second recombination site that interacts with said mutated att recombination site 30 The isolated nucleic acid molecule of claim 29, wherein said mutated att recombination site is a mutated αttL site comprising a core region having the nucleotide sequence caacttnntnnnannaagttg, wherein "n" represents any nucleotide 31 The isolated nucleic acid molecule of claim 30, wherein said mutated αttL recombination site comprises a core region having a nucleotide sequence selected from agcctgctttattatactaagttggcatta (αttL5) and agcctgcttttttatattaagttggcatta (αttL6) 32 The isolated nucleic acid molecule of claim 29, wherein said mutated att recombination site comprises a core region having a nucleotide sequence selected from the group consisting of ggggacaactttgtacaaaaaagttggct (αttB l 6) , ggggacaactttgtacaagaaagctgggt (αttB2.2), and ggggacaactttgtacaagaaagttgggt (αttB2 10) 33 A vector selected from the group consisting of pENTRIA, pEΝTR2B, pENTR3C, pENTR4, pENTR5, pENTR6, pENTR7, pENTR8, pENTR9, pENTRlO, pENTRl l, pDESTl, pDEST2, pDEST3, pDEST4, -174- pDEST5, pDESTό, pDEST7, pDEST8, pDEST9, pDESTl O, pDESTl 1, pDEST12.2 (also known as pDEST12), pDEST13, pDEST14, pDESTl 5, pDESTl 6, pDESTl 7, pDESTl 8, pDESTl 9, pDEST20, pDEST21, pDEST22 , pDEST23, pDEST24, pDEST25, pDEST26, pDEST27, pDEST28, pDEST29, pDEST30, pDEST31, pDEST32, pDEST33, pDEST34, pDONR201 (also known as pENTR21 attP vector or pAttPkan Donor Vector), pDONR202, pDONR203 (also known as pEZ15812), pDONR204, pDONR205, pDONR206 (also known as pENTR22 attP vector or pAttPgen Donor Vector), pDONR207, pMAB58, pMAB62, pMAB85 and pMAB86. 34. A host cell comprising the vector of claim 33. 35. A polypeptide encoded by the vector of claim 33. 36. A kit for use in synthesizing a nucleic acid molecule, said kit comprising the isolated nucleic acid molecule of any one of claims 1-10, 26 and 29. 37. A kit for use in synthesizing a nucleic acid molecule, said kit comprising the primer of claim 15 or claim 18. 38. A kit for use in cloning a nucleic acid molecule, said kit comprising the vector of claim 19 or claim 33.

Inventors: BRASCH MICHAEL A, CHEO DAVID, HARTLEY JAMES L, TEMPLE GARY F, BRASCH, MICHAEL, A., CHEO, DAVID, HARTLEY, JAMES, L., TEMPLE, GARY, F.
Applicants: LIFE TECHNOLOGIES INC, LIFE TECHNOLOGIES, INC.
Application year: 2000
Main classification: C07H21
IPC codes: C12N1, C12N15