Cloning system for construction of recombinant expression vectors

Abstract

Cloning systems are provided for constructing expression vectors. In one aspect of the invention, a kit is provided for constructing one or more recombinant expression vectors. The kit comprises: a linear driver DNA comprising a promoter sequence, a donor recombination site, and at least one selectable marker, the linear driver DNA being capable of being ligated with one or more linear donor DNA comprising a donor DNA sequence to form one or more circular donor DNA; and a circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA to form the recombinant expression vector for expressing the donor DNA sequence.

1 . A library of double-stranded circular donor DNA, comprising: 
 a donor DNA sequence which varies within a library of donor DNA sequences;    a donor recombination site; and    a selectable marker,    wherein the circular donor DNA does not include an origin of replication.    
     
     
         2 . The library of circular donor DNA according to  claim 1 , wherein the library of donor DNA is a library of cDNA or genomic DNA.  
     
     
         3 . The library of circular donor DNA according to  claim 2 , wherein the cDNA library is derived from a single human chromosome.  
     
     
         4 . The library of circular donor DNA according to  claim 1 , wherein the circular donor DNA further comprises a promoter sequence that controls expression of the donor DNA sequence.  
     
     
         5 . The library of circular donor DNA according to  claim 4 , wherein the promoter is derived from bacteria, yeast, insect, animal, plant or virus.  
     
     
         6 . The library of circular donor DNA according to  claim 4 , wherein the promoter is selected from the group consisting of  E. coli  lac and trp operons, the tac promoter, the bacteriophage λ p L  promoter, bacteriophage T7 and SP6 promoters, β-actin promoter, insulin promoter, human cytomegalovirus (CMV) promoter, HIV-LTR, RSV-LTR, SV40 promoter, baculoviral polyhedrin and p10 promoter.  
     
     
         7 . The library of circular donor DNA according to  claim 4 , wherein the promoter is an inducible promoter.  
     
     
         8 . The library of circular donor DNA according to  claim 7 , wherein the inducible promoter is selected from the group consisting of tetracycline, heat shock, steroid hormone, heavy metal, phorbol ester, adenovirus E1A element, interferon, and serum inducible promoters.  
     
     
         9 . The library of circular donor DNA according to  claim 1 , wherein the donor recombination site is a DNA sequence recognized by a site-specific recombinase to facilitate site-specific fusion between the circular donor DNA and an acceptor vector containing an acceptor recombination site.  
     
     
         10 . The library of circular donor DNA according to  claim 1 , wherein the donor recombination site is a recombination site recognized by a recombinase, a transposase or an integrase.  
     
     
         11 . The kit according to  claim 10 , wherein the recombinase is selected from the group consisting of the bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage λ, phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, Hin recombinase, Cin recombinase,  E. coli  xerC and xerD recombinases,  Bacillus thuringiensis  recombinase, TpnI and β-lactamase transposons, and immunoglobulin recombinases.  
     
     
         12 . The library of circular donor DNA according to  claim 1 , wherein the donor recombination site is a Iox site that is recognized by the Cre recombinase of bacteriophage P1.  
     
     
         13 . The library of circular donor DNA according to  claim 12 , wherein the donor recombination site is selected from the group consisting of IoxB, IoxL, IoxR, IoxP, IoxP3, IoxP23, IoxΔ86, IoxΔ117, IoxP511, and IoxC2.  
     
     
         14 . The library of circular donor DNA according to  claim 12 , wherein the donor recombination site is selected from the group consisting of att sites recognized by the Int recombinase of bacteriophage λa, FRT site recognized by FLP recombinase of the 2pi plasmid of  Saccharomyces cerevisiae , a recombination site recognized by the resolvase family, and a recombination site recognized by transposase of  Bacillus thruingiensis.    
     
     
         15 . The library of circular donor DNA according to  claim 1 , wherein the selectable marker is selected from the group consisting of β-galactosidase, fluorescent protein, secreted form of human placental alkaline phosphatase, β-glucuronidase, antibiotic resistance genes, yeast seletable markers leu2-d and URA3, apoptosis resistant genes, and antisense oligonucleotides.  
     
     
         16 . The library of circular donor DNA according to  claim 1 , wherein the circular donor DNA further includes an affinity tag.  
     
     
         17 . The library of circular donor DNA according to  claim 16 , wherein the affinity tag is selected from the group consisting of a polyhistidine tract, polyarginine, glutathione-S-transferase, maltose binding protein, a portion of staphylococcal protein A, protein A, and epitope tag.  
     
     
         18 . The library of circular donor DNA according to  claim 16 , wherein the affinity tag is an EE tag.  
     
     
         19 . The library of circular donor DNA according to  claim 1 , wherein the circular donor DNA further includes a potyadenylation signal.