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Cloning system for construction of recombinant expression vectorsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidCloning system for construction of recombinant expression vectors description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060019291, Cloning system for construction of recombinant expression vectors. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATION [0001] This application is a divisional of U.S. Application entitled "Cloning Systems For Construction of Recombinant Expression Vectors," Ser. No. 10/378,064, filed Feb. 27, 2003 which is a divisional of U.S. Application entitled "Compositions and Methods For Generating Expression Vectors Through Site-Specific Recombination," Ser. No. 09/606,323, filed Jun. 28, 2000, now U.S. Pat. No. 6,551,828. These applications are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION [0002] This invention relates to recombinant DNA technology, nucleic acids, vectors and methods for use in a recombinational cloning or subcloning, and more specifically for constructing expression vectors by using recombination proteins in vitro or in vivo through site-specific recombination. DESCRIPTION OF RELATED ART [0003] Recombinant DNA technology, also called gene cloning or molecular cloning, is widely used to transfer genetic information, i.e. DNA, from one organism to another. A typical recombinant DNA experiment often follows the following procedure. First, the DNA (e.g., the cloned DNA, insert DNA, target DNA, or foreign DNA) from a donor organism is extracted, enzymatically cleaved (or cut/digested), and joined (ligated) to another DNA entity (e.g. a cloning vector) to form a new, recombinant DNA molecule (or cloning vector-insert DNA construct). Second, this cloning vector-insert DNA construct is transferred into and maintained within a host cell, such as transformation of a bacterial host cell by the construct. Third, those host cells that take up the DNA construct (transformed cells) are identified and selected from those that do not. In addition, if required, a DNA construct can be prepared to ensure that the protein product that is encoded by the cloned DNA sequence is produced by the host cell. [0004] Accordingly, this traditional cloning methods using restriction enzymes and ligase can be time consuming, especially when a specific expression vector is required for transferring the target gene into a heterologous host cell, such as a mammalian cell. The specific expression vector may not contain matching restriction sites for the donor DNA. Extensive reengineering of the expression vector may be required to introduce the matching restriction sites into the vector so that the vector and the insert DNA can be ligated to produce the final construct. Alternatively, multiple restriction enzymes may have to be employed to generate an insert DNA having suitable restriction sites for ligation with the vector. In this case, reaction conditions for each restriction enzyme may differ such that it is often necessary to perform a few separate restriction digestion reactions to obtain the desired insert. Further, the efficiency of direct ligation between the vector and insert may be very low, especially between large fragments. As a result, the whole procedure is tedious, and the final yield of the correctly ligated construct can be low. [0005] Site-specific recombination represents another useful method of recombinant DNA technology. This method employs a site-specific recombinase, an enzyme which catalyzes the exchange of DNA segments at specific recombination sites. Site-specific recombinases present in some 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. Landy, A. (1993) Current Opinion in Biotechnology 3:699-707. [0006] A typical site-specific recombinase is Cre recombinase. Cre is a 38-kDa product of the cre (cyclization recombination) gene of bacteriophage P1 and is a site-specific DNA recombinase of the Int family. Sternberg, N. et al. (1986) J. Mol. Biol. 187: 197-212. Cre recognizes a 34-bp site on'the P1 genome called IoxP (locus of X-over of P1) and efficiently catalyzes reciprocal conservative DNA recombination between pairs of IoxP sites. The IoxP site consists of two 13-bp inverted repeats flanking an 8-bp nonpalindromic core region. Cre-mediated recombination between two directly repeated IoxP sites results in excision of DNA between them as a covalently closed circle. Cre-mediated recombination between pairs of IoxP sites in inverted orientation will result in inversion of the intervening DNA rather than excision. Breaking and joining of DNA is confined to discrete positions within the core region and proceeds on strand at a time by way of transient phophotyrosine DNA-protein linkage with the enzyme. Other examples of site-specific recombination systems include the integrase/att system form bacteriophage .lamda. and the FLP/FRT system from the Saccharomyces cerevisiae 2pi circle plasmid. [0007] These site-specific recombination systems have been used in vivo to facilitate recombination between different vectors. Waterhouse et al. used an in vivo method to join light and heavy chains of an antibody. The light and heavy chains were cloned in different phage vectors between IoxP and IoxP 511 sites that were used to transform new E. coli cells. Waterhouse, P. et al. (1993) Nucleic Acid Res. 21:2265-2266. Cre acted on two parental molecules, one plasmid and another phage, in the host cells to produce four products in equilibrium: two different cointegrates (produced by recombination at either IoxP or IoxP511 sites), and two daughter molecules, one of which was the desired product. Schlake and Bode used an in vivo method to exchange expression cassettes at defined chromosomal locations, each flanked by a wild type and spacer-mutated FRT recombination site. Schlake and Bode (1994) Biochemistry 33:12746-12751. A double-reciprocal crossover was mediated in cultured mammalian cells by using the FLP/FRT system for site-specific recombination. Aoki et al. used a shuttle plasmid (pAdMCS) that carried a gene of interest, a IoxP site, the adenoviral 5-LTR and packaging signal 0 to 1 mu, and a multiple cloning site. Aoki et al. (1999) Mol. Med. 5:224-231. The shuttle plasmid was linearized by a restriction enzyme NheI and recombined with ClaI-digested adenoviral cosmid in vitro. Cre recombinase produced the full-length recombinant adenoviral vector in vitro by an exchange of region distal to the IoxP site linearized in these two molecules. SUMMARY OF THE INVENTION [0008] The present invention relates to compositions, kits, and methods for use in a recombinational cloning or subcloning. In particular, the present invention provides novel methods for constructing expression vectors by using site-specific recombinases in vitro. These method may be used for high throughput screening of genes, functional genomics and other human genome projects. [0009] In one aspect, the present invention provides a double-stranded circular donor DNA for transferring a donor DNA sequence into expression vectors. The circular donor DNA comprises: a donor DNA sequence; a donor recombination site; at least one selectable marker, the circular donor DNA not including an origin of replication. [0010] The donor DNA sequence may be any gene of interest or any synthetic DNA sequence which is needed to be transferred into an expression vector. For example the donor DNA segment may be a sequence derived from cDNA of a particular gene or one of the members of a cDNA library. The donor DNA may also be a genomic DNA that contains the coding region interrupted with non-coding sequences. [0011] In another aspect, the present invention also provides a library of double-stranded circular donor DNAs that may be used for high throughput screening. The library of double-stranded circular DNA comprises: a donor DNA sequence which varies within a library of donor DNA sequences; a donor recombination site; and at least one selectable marker, the circular donor DNA not including an origin of replication. [0012] The library of donor DNA sequences may be a library of cDNA or genomic DNA derived from any desirable sources. For example, the library of donor DNA sequences may be a cDNA library from single human chromosomes. [0013] The circular donor DNA may further comprise a promoter sequence that controls expression of the donor DNA sequence. The promoter may be any array of DNA sequences that interact specifically with cellular transcription factors to regulate transcription of the downstream gene. The promoter may be derived from any organism, such as bacteria, yeast, insect and mammalian cells and viruses. Examples of the promoter include, but are not limited to, E. coli lac and trp operons, the tac promoter, the bacteriophage .lamda. p.sup.L promoter, bacteriophage T7 and SP6 promoters, .beta.-actin promoter, insulin promoter, human cytomegalovirus (CMV) promoter, HIV-LTR (HIV-long terminal repeat), Rous sarcoma virus RSV-LTR, simian virus SV40 promoter, baculoviral polyhedrin and p10 promoter. [0014] The promoter may also be an inducible promoter that regulates the expression of downstream gene in a controlled manner. Examples of inducible promoters include, but are not limited to, the bacterial dual promoter (activator/repressor expression system) which regulates gene expression in mammalian cells under the control of tetracycline and its analogs and promoters that regulate gene expression under the control of factors such as heat shocks, steroid hormones, heavy metals, phorbol ester, the adenovirus E1A element, interferon, or serum. [0015] The donor recombination site may be any segment or arrays of DNA sequence recognized by a site-specific recombinase which catalyzes site-specific fusion between the circular donor DNA and an acceptor vector. The site-specific recombinase may be a recombinase, a transposase or an integrases. [0016] In one variation, the recombination site is a Iox site that is recognized by the Cre recombinase of bacteriophage PI. Example of Iox site includes, but are not limited to, IoxB, IoxL, IoxR, IoxP [SEQ ID NO:1], IoxP3, IoxP23, Iox.DELTA.86, Iox.DELTA.117, IoxP511 [SEQ ID NO:2], and IoxC2 [SEQ ID NO:3]. [0017] In another variation, the recombination site is a recombination site that is recognized by a recombinases other than Cre. Examples of the non-Cre recombinases include, but are not limited to, site-specific recombinases include: att sites recognized by the Int recombinase of bacteriophage .lamda. (e.g. att1, att2, att3, attP, attB, attL, and attR), the FRT sites recognized by FLP recombinase of the 2pi plasmid of Saccharomyces cerevisiae, the recombination sites recognized by the resolvase family, and the recombination site recognized by transposase of Bacillus thruingiensis. [0018] The example of site-specific recombinase include, but are not limited to, bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage .lamda., phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, the Hin recombinase, and the Cin recombinase, E coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, TpnI and the .beta.-lactamase transposons, and the immunoglobulin recombinases, [0019] The selectable marker of the circular donor DNA may be any functional element for facilitating subsequent identification and selection of clones of the recombination product under suitable conditions. The selectable marker may encode any functional element, such as protein, peptide, RNA, binding site for RNA and proteins, or products that provide resistance to organic or inorganic agents. Examples of selectable markers include, but are not limited to, reporter genes such as .beta.-galactosidase (GAL), fluorescent proteins (e.g., GFP, GFP-UV, EFFP, BFP, EBFP, ECFP, EYFP), secreted form of human placental alkaline phosphatase (SEAP), .beta.-glucuronidase (GUS)); resistance genes against antibiotics (e.g. neomycin (G418) or hygromycin resistant gene, puromycin resistant gene), yeast seletable markers leu2-d and URA3, apoptosis resistant genes (e.g. baculoviral p35 gene), and antisenoligonucleotides. [0020] The circular donor DNA may optionally include an affinity tag for selection and isolation of protein product encoded by the donor DNA segment. Examples of such an affinity tag include, but are not limited to, a polyhistidine tract, polyarginine, glutathione-S-transferase (GST), maltose binding protein (MBP), a portion of staphylococcal protein A (SPA), and various immunoaffinity tags (e.g. protein A) and epitope tags such as those recognized by the EE (Glu--Glu) antipeptide antibodies. The affinity tag may be positioned at either the amino- or carboxy-terminus of the donor DNA. Continue reading about Cloning system for construction of recombinant expression vectors... Full patent description for Cloning system for construction of recombinant expression vectors Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Cloning system for construction of recombinant expression vectors patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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