Construction of a λ Packageable ColEl Vector which Permits Cloning of Large DNA Fragments: Cloning of thyA Gene of Escherichia coli

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Abstract
A small packageable plasmid depending on the ColEl replicon was constructed from CoXEl-gal-cosλ::-.Tn3, pKY2113 (T. Nakamura et al. (1981) J. Biochem. 90, 1013), and named pKY2662. This plasmid carriesampicillin resistance and cohcin El 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, BglII, ClaI, EcoRI, HpaI, PstI, and Tth11II. 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.