pCGS990

Conversion vector to insert copy number control elements into existing pYAC4-derived clones by targeted homologous recombination. pCGS990 should be linearized with SalI before transformation of the YAC-carrying strain and Lys+ clones selected. Homologous recombination between pCGS990 and the target YAC results in replacement of the centric pYAC4 arm with sequences from pCGS990. Recombinant clones will be Trp-. Approximately 0.5-2.5 percent of transformants will have the desired phenotype (Lys+, Ura+, Trp-). These clones may be grown in liquid MST medium for 2-5 days to increase the YAC copy number. The cis-acting elements necessary for amplification are the thymidine kinase gene and a conditional CEN4 regulated by GAL1. The order of the major features in the plasmid is: BamHI – Tetrahymena telomere – ampR – thymidine kinase – EcoRI – LYS2 – ARS1 – GAL1 promoter – CEN4 – ClaI – SalI. [1] Medium is LB plus ampicillin (ATCC medium number 1227) 37C Deposited by: D.T.Moir Hosts: E.coli HB101, Saccharomyces cerevisiae.