Two cat expression vectors for cloning and generation of 3'- and 5'-deletion mutants

The construction of two versatile cat vectors, pGK0CAT and pGKA10CAT, is reported. These vectors possess multiple cloning sites derived from the Bluescript pKS(+) plasmid that allow the cloning of diverse DNA fragments. From a single cloned insert, i.e., a putative promoter element, one can use the exonuclease III (ExoIII) and S1 or mung-bean nuclease method to generate sequential deletion mutants of the 3' and 5' region. Linker-scanning and internal deletion mutants can thus be created by using appropriate 3'- and 5'-deletion mutants. These plasmids could thus be used for the identification of cis-acting promoter or enhancer elements by either in vivo or in vitro transcriptional analyses. The ability of these vectors to generate deletion mutants from the 3' end make them suitable to identify cis-acting elements in the 5'-noncoding region of the mRNA involved in the translational regulation of protein synthesis. Single-stranded circular mutant plasmids could also be generated from these vectors to study various protein-DNA interactions.