pCGS990 [CGE1684] (ATCC® 77417)

Applications: YR-type (replicating) shuttle vectorshuttle vectorvector permitting positive selection for integration  /  Depositors: DT Moir

Permits and Restrictions

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Designations pCGS990 [CGE1684]
Depositors DT Moir
Biosafety Level 1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Vector Information
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
centromere: CEN4
YR-type (replicating) shuttle vector
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.
Media ATCC® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Growth Conditions
Temperature: 37.0°C

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

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