Restriction digests of the clone give the following sizes (kb): EcoRI--14.0; BamHI--10.0, 4.4; XbaI--14.0. 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-. The cis-acting elements necessary for amplification are the thymidine kinase gene and a conditional CEN4 regulated by GAL1. 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. Conversion vector to insert copy number control elements into existing pYAC4-derived clones by targeted homologous recombination. The order of the major features in the plasmid is: BamHI - Tetrahymena telomere - ampR - thymidine kinase - EcoRI - LYS2 - ARS1 - GAL1 promoter - CEN4 - ClaI - SalI. |
