US 6,551,828 B1Granted

Compositions and methods for generating expression vectors through site-specific recombination

AssigneeProtemation, Inc.

InventorRobin Clark

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Abstract

Compositions, kits, and methods are provided for use in a recombinational cloning or subcloning methods for constructing expression vectors which comprise: ligating a library of double-stranded linear donor DNAs, where each member of the library includes a donor DNA sequence, with a double-stranded linear driver DNA which includes a promoter sequence and a donor recombination site to form a single circular donor DNA, the single circular donor DNA not including an origin of replication, where the donor DNA sequence is under the transcriptional control of the promoter; and contacting the circular donor DNA and a circular acceptor acceptor vector in the presence of a recombinase to form a single fused circular vector, the circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA.

Claims28

Dependent claims

A method for generating a recombinant expression vector for expressing a donor DNA sequence, comprising:

contacting a circular double-stranded donor DNA and a circular acceptor vector in the presence of a recombinase under conditions suitable for the circular double-stranded donor DNA and the circular acceptor vector to recombine to form a single fused circular vector that combines sequences of both the circular double-stranded donor DNA and the circular acceptor vector, thereby forming the recombinant expression vector for expressing the donor DNA sequence, wherein
the circular double-stranded donor DNA comprises a donor DNA sequence, a single donor recombination site, and at least one selectable marker, but not including an origin of replication;
the circular acceptor vector comprises an origin of replication and a single acceptor recombination site capable of recombining with the circular donor DNA;
and
the recombinase recognizes the donor and acceptor recombination sites and catalyzes fusion of the whole sequence of the circular double-stranded donor DNA into the circular acceptor vector.

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Dependencies are inferred from the claim text.