Direct selection shuttle plasmid vector, pPW264, used for cloning the alpha-amylase gene of Schwanniomyces occidentalis.

Abstract

We constructed a novel cloning system with positive selection for inserted fragments. The gene for tetracycline resistance (tetR) originally used in plasmid pTR262 was replaced with the gene for chloramphenicol acetyltransferase (cat) and terminator sequences were introduced downstream of the cat gene. The terminator sequences stop transcription originating on strong PR promoter that would otherwise proceed through the region of replication origin and interfere with plasmid replication. Thus the copy number of recombinant plasmid molecules is stabilized. The cloning system has been constructed in a new YEp type shuttle vector, pPW264. The 8.1 kb-vector carries two unique cloning sites, BglII and HindIII. The maintenance of the vector and selection in yeast is ensured by URA3 Saccharomyces cerevisiae gene. The vector was employed in cloning of the gene for alpha-amylase from Schwanniomyces occidentalis.