Gene, 50 (1986) 3-40 Elsevier GEN 01758 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Plasmid vector pBR322 and its s~ i~ -~ ~ ~ derivatives - a review (Recombinant DNA; EK2 multipurpose cloning vehicles; nucleotide sequence) Pauliaa Balbhs, Xavier Soberh, Enrique Merino, Mario Zurita, H.ilda Lomeli, Fernando Valle, Noemi Flares and Francisco Bolivar * zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Centro de Investigation sobre Ingenieria Genktica y Biotecnologia, Universidad Naeional Asthma de M ~tico, Cuernavuca, Morelos {M&co) Tel. f52?3)1 Y- 23- 99 (Received August 26th 1985) (Accepted September lst, 1986) SUMMARY The plasmid pBR322 was one of the fast EK2 m~tip~ose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichiu co&. This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence. The widespread use of pBR322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmid’s effectiveness as a cloning vector: (a) plasmid instabihty in the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO absence of seiection and, (b) the lack of a direct selection scheme for r~ornb~~t DNA molecules. Several vectorsbased on pBR322 have been ~ons~ct~ to overcome these limitations and to extend the vector’s versatility to accomodate special cloning purposes. The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322. INTRG~U~ION Since the early days of molecular cloning, bacterial plasmids have received much attention because of their usefulness as vectors in recombinant DNA experimentation. The design and construction of cloning vectors has now become a complex and highly sop~s~~at~ area of study, yielding a vast amount of ~fo~a~on and a great number of spe- cialiied vectors for investigators to use. Part of our group has been involved in the devel- opment of a series of multipurpose cloning vehicles. One ofthese vectors, pBR322 (Bolivar et al., 1977b), continues to be the most widely used cloning vehicle * To whom ~o~es~ndence and reprint requests should be addressed at Apartado P&al 70479, Mexico 04510 DF (Mexico). Abbreviations: aa, amina acid(s); AGPT, amino glycosyl 3’ phosphotransferase gene from Tn5; Ap, ampicillin;ARS, autono- mous replication sequence; jIGal, bgalactosidase; born, basis of mobiiation; bp, base pair(s); CAT, Cm acetyl-transferase; Cb, c~~c~~n; Cm, ~hlor~phenicol; Gm, gent~yc~; IPTG, iso- prowl-~-~~og~actop~~oside; kb, 1000 bp; Km, k~~yci~; Neo, neomycin; nt, nucleoti~s); put, partition locus; ti, DNA replica&r origin; rep, repressor of primer; R, resistance; RRS, ribosome~binding site; SCP, synthetic consensus promoter; Sm, streptomycin; Sp, spectinomycin; Su, sulfonamide; ss, single strandled); Tc, tetracycline; Thicr, tbiostrepton; US, unique sites; Vio, viomycin; XGal, 5-b~m~chloro-indoyl-8_D-galac- toside.