Plasmid vehicles for direct cloning of Escherichia coli promoters

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Abstract
A multicopy plasmid cloning vehicle, pGA22, which carries genes for ampicillin resistance (Apr), tetracycline resistance (Tcr), chloramphenicol resistance (Cmr) and kanamycin resistance (Kmr) was constructed. This plasmid has 5 unique sites for restriction endonucleases EcoRI, PstI, XhoI, SmaI and SalI within antibiotic resistance genes. pGA22, which is 5.1 megadaltons in size, has a low copy number (probably fewer than 10/genome), is capable of relaxed replication and is mobilized by F-factor at a frequency of 10-5. A series of promoter-cloning vehicles, pGA24, pGA39 and pGA46, was developed from pGA22. In these plasmids the natural promoter for Tcr was removed and replaced by small DNA fragments carrying unique sites for several restriction endonucleases. Cells carrying these vectors are sensitive to tetracycline unless insertional activation of the Tcr occurs by cloning a promoter-carrying DNA fragment in 1 of the unique sites adjacent to the 5'' end of Tcr. In this way, promoters carried on a HindIII-generated DNA fragment can be inserted at the HindIII site of plasmid pGA24, pGA39, or pGA46. A promoter in fragments generated by digestion with restriction endonuclease XmaI or PstI or by any restriction endonucleases which generate flush ends, such as SmaI, PvuII, HpaI, HincII, or HaeIII, can be cloned in plasmid pGA39. Plasmid pGA46 can be used to detect a promoter fragment carried on a BglII, BamHI, MboI, or PstI fragment. A plasmid, pGA44, was described with a unique kpnI site in the rifampin resistance gene rpoB.