Size (kb): 5.6999998092651370
Vector: pUN65 (plasmid)
Promoters: Promoter lac
Construction: pUC18, pBluescript KS+
Construct size (kb): 5.699999809265137
Features: insert detection: lacZ'
promoter for in vitro transcription: T3
promoter for in vitro transcription: T7
YC-type (centromeric) shuttle vector
vector permitting RNA synthesis in vitro
vector permitting visual detection of recombinants beta-galactosidase beta-D-galactosidase
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)
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.
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