In vivo plasmid construction by integrative recombination within a 156 bp sequence homology

pHG165, a pBR322 copy number derivative of pUC8, has a 38-bp polylinker multiple cloning site located at the 5' end of lac alpha. A further 156 bp, 3' to the multiple cloning site, completes the coding sequence for the production of the beta galactosidase alpha peptide. We describe the use of in vivo plasmid-chromosome cointegrates as a construction method that in this instance has enabled us to cross out the pHG165 multiple cloning site to obtain a wild-type beta-galactosidase sequence for the alpha peptide. Because the DNA sequence available for homologous recombination was only 156 bp in length, the frequency of crosses that removed the multiple cloning site was less than 1 x 10(-9). These crosses were easily obtained, however, after amplification by ampicillin selection.