Size (kb): 14.0000000000000000
Vector: pCGS990 (plasmid)
Promoters: Promoter GAL1
Construction: LYS2, CEN4, pBR322, pCGS986, pYTCa-1
Construct size (kb): 14.0
Features: marker(s): ampR, LYS2
promoter: GAL1, HSV TK
replicon: pMB1, ARS1
YR-type (replicating) shuttle vector
vector permitting positive selection for integration
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.
Smith DR, et al. Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination. Mamm. Genome 4: 141-147, 1993. PubMed: 8439726