DIRECT SELECTION SHUTTLE PLASMID VECTOR, PPW264, USED FOR CLONING THEALPHA-AMYLASE GENE OF SCHWANNIOMYCES-OCCIDENTALIS

We constructed a novel cloning system with positive selection for inse rted fragments. The gene for tetracycline resistance (tet(R)) original ly used in plasmid pTR262 was replaced with the gene for chloramphenic ol acetyltransferase (cat) and terminator sequences were introduced do wnstream 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 replicati on. Thus the copy number of recombinant plasmid molecules is stabilize d. The cloning system has been constructed in a new YEp type shuttle v ector, pPW264. The 8.1 kb-vector carries two unique cloning sites, Bgl II and HindIII. The maintenance of the vector and selection in yeast i s ensured by URA3 Saccharomyces cerevisiae gene. The vector was employ ed in cloning of the gene for a-amylase from Schwanniomyces occidental is.