Sequence specific recombinase-based methods for producing intron containing vectors and compositions for use in practicing the same

Abstract

Methods are provided for producing a vector that includes at least one splicable intron. In the subject methods, intron containing vectors are produced from donor and acceptor vectors that each include a site specific recombinase site, where the subject donor and acceptor vectors further include splice donor and acceptor sites that, upon site specific recombination of the donor and acceptor vectors, define an intron in the product vector of the recombination step. Also provided are compositions for use in practicing the subject methods, including the donor and acceptor vectors themselves, as well as systems and kits that include the same. The subject invention finds use in a variety of different applications, including the production of expression vectors that encode C-terminal tagged fusion proteins, the production of expression vectors that encode pure protein and not a fusion thereof, and the like.

1 - 11 . (canceled)  
     
     
         12 . A donor vector comprising: 
 (a) at least one recombinase recognition site; and    (b) a splice sequence.    
     
     
         13 . The donor vector according to  claim 12 , wherein said donor vector comprises first and second recombinase recognition sites oriented in the same direction and flanking a portion of a selectable marker, wherein said first and second recombinase recognition sites are able to recombine with each other  
     
     
         14 . The donor vector according to  claim 12 , wherein said donor vector further comprises a coding sequence for a protein of interest.  
     
     
         15 . The donor vector according to  claim 14 , wherein said donor vector is a plasmid, cosmid, bac, yac or virus.  
     
     
         16 . An acceptor vector comprising: 
 (a) at least one recombinase recognition site; and    (b) a splice sequence.    
     
     
         17 . The acceptor vector according to  claim 16 , wherein said recombinase recognition sites are selected from the group consisting of: lox sites, att sites, dif sites and frt sites.  
     
     
         18 . The acceptor vector according to  claim 16 , wherein said recombinase recognition site is a lox site.  
     
     
         19 . The acceptor vector according to  claim 16 , wherein said acceptor vector further comprises an origin of replication.  
     
     
         20 . The acceptor vector according to  claim 19 , wherein said acceptor vector is a plasmid, cosmid, bac, yac or virus.  
     
     
         21 - 30 . (canceled)  
     
     
         31 . An intron containing vector comprising: 
 (a) at least one recombinase recognition site; and    (b) a spliceable intron.    
     
     
         32 . The vector according to  claim 31 , wherein said vector comprises first and second recombinase recognition sites oriented in the same direction;  
     
     
         33 . The vector according to  claim 32 , wherein said vector further comprises: 
 an expression cassette for a protein of interest divided into two subparts that flank said first recombinase recognition; and    a functional marker divided into two sub-parts that flank said second recombinase recognition site.    
     
     
         34 . The vector according to  claim 31 , wherein said recombinase recognition sites are selected from the group consisting of: lox sites, att sites, dif sites and frt sites.  
     
     
         35 . The vector according to  claim 34 , wherein said recombinase recognition sites are lox sites.  
     
     
         36 . The vector according to  claim 31 , wherein said vector is a plasmid, cosmid, bac, yac or virus.  
     
     
         37 . A nucleic acid library cloned into a plurality of vectors selected from the group consisting of donor vectors according to  claim 12  and acceptor vectors according to  claim 16.