Construction of vectors with the pl5a replicon, kanamycin resistance, induciblelacZα and pUC18 or pUC19 multiple cloning sites

Complementation analysis is a powerful technique in the characterization of genes and their products. When studying cloned genes the analysis is facilitated if compatible vectors are available into which restriction fragments can be cloned. Most multicopy cloning vectors currently in use are derivatives of pBR322 (1), with ampicillin (Ap) resistance markers and the ColEl origin of replication. Vectors such as pUC18, pUC19 (2) and the pBluescript vectors (e.g. pKS-) (3) also contain a multiple cloning site (MCS) in a lacZa gene fragment for ease of selection of inserts on X-Gal plates, and controlled expression of insert genes using IPTG. We have developed a pair of compatible vectors, with a kanamycin (Km) resistance marker, the pl5a origin of replication, and the lacZa gene fragment, for use in complementation studies with ColEl-based clones. The pK18 and pK19 vectors described by Pridmore (4) were used as the basis for the new vectors. These are pUC based vectors with ColEl replicons and a Km resistance gene replacing the Ap resistance gene of the pUC series. The ColEl origin was removed by partial digestion with NspHl and the linearized plasmid was isolated, digested with BstBl and blunt ended with T4 DNA polymerase and dNTPs. We used pACYC184 (5) as the source for the pi5a origin of replication. It was removed as a 685 bp Sacll-Clal fragment and treated with T4 DNA polymerase and dNTPs to create a blunt-ended fragment. The