Patent ID: 7087415

Claim:
A method for generating a population of variant DNA molecules in bacterial cells, said method comprising: (a) transferring a donor vector into a bacterial cell capable of homologous recombination, wherein (i) said donor vector comprises a donor recombination module comprising, in the following order from 5′ to 3′: a first donor DNA sequence and a second donor DNA sequence, and (ii) said bacterial cell comprises a target vector comprising a target recombination module comprising, in the following order from 5′ to 3′: a first target DNA sequence; a negatively selectable marker; and a second target DNA sequence, wherein said first donor DNA sequence is homologous to said first target DNA sequence, and said second donor DNA sequence is homologous to said second target DNA sequence, wherein at least one of (1) the first donor DNA sequence and the first target DNA sequence are not identical to each other, or (2) the second donor DNA sequence and the second target DNA sequence, are not identical to each other; and (b) selecting for a population of bacterial cells within which homologous recombination between the donor vector and the target vector has occurred, such that the cells do not contain the negatively selectable marker, thereby generating a population of variant DNA molecules in bacterial cells.