An improved positive selection plasmid vector constructed by ollgonucleotide mediated mutagenesis

摘要

An Escherichia coli plasmid vector, pUN121, has been constructed which allows for positive selection of transformants harboring DNA inserts. The vector is based on plasmid pTR262 (Roberts et al, Gene, 12, (1980), 123–127) in which the tetracycline resistance gene is under transcriptional control of the repressor protein coded by the phage lambda cI gene. This plasmid has been rearranged, using in vitro recombinant techniques including oligonucleotide mediated mutagenesis to yield a smaller plasmid (4.4 kb) with unique cloning sites for EcoRI, XmaI and SmaI in addition to the unique HindHI and Bell sites. The plasmid has a functional ampicillin resistance gene and the new restriction sites (EcoRI, XmaI and Smal) when used for cloning, give rise to tetracycline resistant transformants.