Specific-purpose plasmid cloning vectors I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors
1981
Abstract Two cloning vector plasmids, pHSG415 (7100 bp) and a λ phage cos site-containing derivative (cosmid) thereof, pHSG422 (8760 bp), were constructed from a low copy number plasmid (pSC101) replicon to permit the propagation of cloned DNA segments at low gene dosage levels. Two features of the vectors, namely temperature sensitivity of replication and inability to be mobilized by conjugative plasmids, cause them to exhibit a high level of “biological containment”. The essential characteristics of pHSG415 and pHSG422 may be summarized as follows: (1) their genome copy number is low (4–6 copies/chromosome); (2) their replication ceases at high temperature and they are rapidly lost from host cells grown at temperatures of 37°C and above; (3) the relaxation nick site of pSC101, which is thought to be synonymous with its origin of transfer replication, is absent from the vectors; as a consequence, they are not mobilized to a significant extent by co-existing conjugative plasmids that are able to mobilize wild-type pSC101; (4) they contain unique insertion sites for DNA fragments generated by the following restriction endonucleases: Eco RI, Xho I, Xma I, Hin dIII and Pst I; pHSG415 additionally contains single Bam HI, Bst EII and Hin cII sites and may also be used to clone Pvu I-generated fragments; (5) the plasmids confer upon their host cells resistance to chloramphenicol, kanamycin and ampicillin, and every unique cloning site, except those of Bam HI and Bst EII, is located within one of these antibiotic-resistance genes.
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