Gene, 18 (1982) 335-341 Elsevier Biomedical Press 335 zyxwvutsr Versatile low-copy-number plasmid vectors for cloning in Escherichia coli (Recombinant DNA; pSC101, pSClO5; pLG338, pLG339; tetracycline, kanamycin resistance; phage x pL promoter) Neil G. Stoker, Neil F. Fairweather * and Brian G. Spratt ** Department of Genetics, University of Leicester, Leicester LEI 7RH; * zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR W ellcome Research Laboratories, Beckenham, Kent, and ** Microbial Genetics Group, University of Sussex, Falmer, Brighton (U.K.) (Received February 19th, 1982) (Accepted April 2Oth, 1982) SUMMARY Small low-copy-number plasmid vectors were constructed by in vitro and in vivo recombinant DNA techniques. pLG338 and pLG339 are derived from pSC105, have a copy number of six to eight per chromosome, and carry genes conferring resistance to tetracycline and kanamycin. pLG338 (7.3 kb) has unique restriction endonuclease sites for BumHI, SalI, HincII, SmaI, XhoI, EcoRI and KpnI, the first five lying within a drug resistance gene. pLG339 (6.2 kb) lacks the KpnI site, but has unique QhI and PuuII sites. These versatile vectors should be useful for cloning many genes coding for membrane and regulatory proteins which cannot be cloned into high-copy-number plasmids. INTRODUCTION Multicopy plasmids are the most commonly used vectors for cloning in Escherichia coli. Most of these vectors are based on the replicons of ColEl, pMB1 or P15A, have copy numbers rang- ing from 18 to 60 per chromosome and can be amplified in the absence of protein synthesis. High-copy-number vectors aid the isolation of large amounts of plasmid DNA and also help in the Abbreviations: ApR, resistance to ampicillin; CmR, resistance to chloramphenicol; DTT, dithiothreitol; e.o.m., efficiency of mobilization; kb, kilobase; KmR, resistance to kanamycin; TcR, resistance to tetracycline; Tc’, sensitivity to tetracycline; [ 1, indicates plasmid-carrier state. purification of proteins coded by cloned genes. There are, however, occasions when a high copy number is unsuitable. Several examples have been reported of genes which have not been successfully cloned onto a high-copy-number vector, but which have been cloned into bacteriophage h, or a plas- mid with a low copy number. These include polA (Murray and Kelley, 1979), dnuA (Hansen and von Meyenburg, 1979), ompA (Beck and Bremer, 1980) and dacA (Spratt et al., 1980; and unpublished results). It seems likely that multiple copies of many genes coding for regulatory or membrane proteins will be deleterious to the cell. Further- more, attempting to clone these genes onto a mul- ticopy plasmid may result in the cloning of a mutant gene. 0378-I 119/82/0000-0000/$02.75 0 1982 Elsevier Biomedical Press