pUN65 [pNN345] (ATCC® 77267)

Applications: YC-type (centromeric) shuttle vectormutation detectionshuttle vectorvector permitting RNA synthesis in vitrovector permitting visual detection of recombinants beta-galactosidase beta-D-galactosidase  /  Depositors: SJ Elledge

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Designations pUN65 [pNN345]
Depositors SJ Elledge
Biosafety Level 1
Vector Information
Size (kb): 5.6999998092651370
Vector: pUN65 (plasmid)
Promoters: Promoter lac
Construction: pUC18, pBluescript KS+
Marker(s):URA3,ampR,SUP11
Construct size (kb): 5.699999809265137
Features: insert detection: lacZ'
marker(s): SUP11
marker(s): URA3
marker(s): ampR
promoter: lac
promoter for in vitro transcription: T3
promoter for in vitro transcription: T7
replicon: ARS1
replicon: pMB1
MCS: KpnI...SacI
centromere: CEN4
Applications
YC-type (centromeric) shuttle vector
mutation detection
shuttle vector
vector permitting RNA synthesis in vitro
vector permitting visual detection of recombinants beta-galactosidase beta-D-galactosidase
Comments
Restriction digests of the clone give the following sizes (kb): XhoI--5.8; EcoRI--5.8; NotI--5.8.
SUP11 in the vector suppresses the red colony phenotype of ade2-101 ochre allele host strains, grown on limiting adenine media. In the absence of plasmid selection, plasmids are lost at a frequency of 1 to 3%, giving rise to red/white sectoring.
One strategy for mutation scanning is to clone the normal allele of a gene into a SUP11 vector and transform into an ade2-101 host with a null mutation in the same gene, thus selecting for plasmid maintenance and producing white colonies.
Vectors pUN20 (ATCC 77263), pUN25 (ATCC 77264), pUN60 (ATCC 77266) and pUN65 (ATCC 77267) contain SUP11.
A second, potentially mutated copy of the gene can then be cloned into a second centromeric shuttle vector (pUN30, ATCC 77265; pUN70, ATCC 77268; pUN90, ATCC 77269; pUN100, ATCC 77270) for transformation into the SUP11 plasmid containing host.
A normal gene in plasmid #2 can relieve the selective pressure on the SUP11 plasmid, restoring the red/white sectoring; a null allele in plasmid #2 will result in SUP11 plasmid maintenance and white colonies.
YC-type shuttle vector useful for the sectoring-shuffle mutagenesis assay. Contains promoters for in vitro RNA synthesis and permits visual detection of recombinants by lacZalpha complementation.
Media ATCC® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Growth Conditions
Temperature: 37.0°C
References

Elledge SJ, Davis RW. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene 70: 303-312, 1988. PubMed: 3063604

Shipped freeze-dried
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