Rapid subcloning using site-specific recombination
AssigneeBaylor College of Medicine
InventorsStephen J. Elledge, Qinghua Liu
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Abstract
The present invention provides compositions, including vectors, and methods for the rapid subcloning of nucleic acid sequences in vivo and in vitro. In particular, the invention provides vectors used to contain a gene of interest that comprise a sequence-specific recombinase target site. These vectors are used to rapidly transfer the gene of interest into any expression vector that contains a sequence-specific recombinase target site located downstream of a promoter element so that the gene of interest may be expressed.
Claims24
A pUNI vector construct comprising, in 5' to 3' operable order:
- a) a conditional origin of replication;
- b) a sequence-specific recombinase target site having a 5' and a 3' end;
- and
- c) a unique restriction enzyme site, said restriction enzyme site located adjacent to said 3' end of said sequence-specific recombinase target site.
A pUNI vector construct comprising, in 5' to 3' operable order:
- a) a conditional origin of replication;
- b) a sequence-specific recombinase target site;
- and
- c) a polylinker, said polylinker located adjacent to said 3' end of said sequence-specific recombinase target site.
A pHOST expression vector construct comprising in 5' to 3' operable order:
- a) an origin of replication;
- b) a promoter element;
- and
- c) a sequence-specific recombinase target site.
A fused expression vector construct produced by the recombination of a pUNI vector and a pHOST expression vector, said fused expression vector comprising in 5' to 3' operable order:
- a) a promoter element;
- b) a first sequence-specific recombinase target site having a 5' and a 3' end;
- c) a gene of interest joined to said 3' end of said sequence-specific recombinase target site such that a functional translational reading frame is created;
- d) a conditional origin of replication;
- e) a first selectable marker gene;
- f) a second sequence-specific recombinase target site;
- and
- g) an origin of replication.
A method for the in vitro recombination of a pUNI vector and a pHOST expression vector to form a fused expression vector, said method comprising:
- a) providing:
- i) a pUNI vector construct comprising, in 5' to 3' operable order, a conditional origin of replication, a first sequence-specific recombinase target site and a first selectable marker gene;
- ii) a pHOST expression vector construct comprising, in 5' to 3' operable order, an origin of replication, a promoter element and a second sequence-specific recombinase target site;
- and
- iii) a site-specific recombinase;
- b) contacting said pUNI vector and pHOST expression vector constructs in vitro with said site-specific recombinase under conditions such that said first and second constructs are recombined to form a single fused expression vector.
A method for the recombination of pUNI vector and pHOST expression vector constructs to form a single fused expression vector in a prokaryotic host, comprising:
- a) providing:
- i) a pUNI vector construct comprising a conditional origin of replication, a first sequence-specific recombinase target site having a 5' and a 3' end, a unique restriction enzyme site, said restriction enzyme site located adjacent to said 3' end of said sequence-specific recombinase target site, and a first selectable marker gene;
- ii) a pHOST expression vector construct comprising in 5' to 3' operable order, an origin of replication, a promoter element having a 5' and a 3' end and a second sequence-specific recombinase target site having a 5' and a 3' end;
- iii) a prokarvotic host cell expressing a site-specific recombinase;
- b) introducing said pUNI vector and pHOST expression vector constructs into said prokaryotic host cell, wherein said host cell expresses a site-specific recombinase, under conditions such that said pUNI vector and pHOST expression vector constructs are recombined to form a single fused expression vector.
Dependencies are inferred from the claim text.