Construction of a lambda packageable ColE1 vector which permits cloning of large DNA fragments: cloning of thyA gene of Escherichia coli.

A small packageable plasmid depending on the ColE1 replicon was constructed from ColE1-gal-cos lambda:: Tn3, pKY2113 (T. Nakamura et al. (1981) J. Biochem. 90, 1013), and named pKY2662. This plasmid carries ampicillin resistance and colicin E1 immunity genes as selective markers, and has neither mobilization function nor movable transposon. The molecular size of pKY2662 is 8.7 kb, and it has a single cleavage site each for BamHI, Bg/II, ClaI, EcoRI, HpaI, PstI, and Tth111I. By using pKY2662 as a vector, a 32 kb Escherichia coli DNA fragment covering thyA, recC, recB, and argA genes was cloned. This new small cosmid is among the most efficient vectors hitherto found for in vitro cloning of large DNA fragments.