pRS401 plasmid in E. coli (ATCC® 87473)

Applications: YI-type (integrating) shuttle vectorvector permitting visual detection of recombinants O-acetylhomoserine sulfhydrylase  /  Depositors: JD Boeke

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Designations pRS401 plasmid in E. coli
Depositors JD Boeke
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.

Distribution host: Escherichia coli HB101 (ATCC 33694)
Vector Information
Size (kb): 4.893
Intact vector size: 4.893
Type of vector: phagemid
Cloning sites: SacI BstXI SacII EagI NotI SpeI BamHI SmaI PstI ClaI SalI
XhoI ApaI KpnI
Polylinker sites: SacI BstXI SacII EagI NotI XbaI SpeI BamHI SmaI PstI EcoRI
EcoRV HindIII ClaI SalI XhoI ApaI KpnI
Construction: pJK142
Host range: Saccharomyces cerevisiae; Escherichia coli
Features (with orientation and position when available):
marker(s): MET15, ->, 421-1270
replicon: f1, <-, 1860-2316
promoter for in vitro transcription: T7, ->, 2486-2505
MCS: KpnI...SacI, ->, 2513-2619
promoter for in vitro transcription: T3, <-, 2632-2651
promoter: lac, <-, 2721-2749
coding sequence: lacZ', <-, 2317-2676
replicon: pMB1, 3075-3075
marker(s): ampR, <-, 3833-4693
Vector: pRS401 (phagemid)
Promoters: Promoter for in vitro transcription T7
Construction: pJK142
Construct size (kb): 4.893
Features: marker(s): MET15
marker(s): ampR
promoter: lac
promoter for in vitro transcription: T3
promoter for in vitro transcription: T7
replicon: f1
replicon: pMB1
MCS: KpnI...SacI
coding sequence: lacZ'
YI-type (integrating) shuttle vector
vector permitting visual detection of recombinants O-acetylhomoserine sulfhydrylase
Restriction digests of the clone give the following sizes (kb): BamHI--4.9; EcoRI--3.2, 1.7; XbaI--2.9, 2.0.
met15 phenotype produces brown colonies when grown on Pb containing media.
This requires two approx. 60nt PCR primers; the 20nts of sequence at the 3' ends of each primer is specific for amplifying the MET15 gene from pRS401, and the 40nts of sequence at the 5' ends matches the genomic sequences flanking the gene of interest.
These same primers can be used to amplify an ADE2 marker gene disruption product from pRS402 (ATCC 87477).
MET15, MET17 and MET25 are synonymous.
pRS401 can be used to generate a gene specific MET15 marker gene disruption cassette for tranformation in gene knockout experiments.
The 20nt PCR primer sequences for generating the MET15 marker from pRS401 are: 5'-CTGTGCGGTATTTCACACCG-3' (left primer) and 5'-AGATTGTACTGAGAGTGCAC-3' (right primer).
Media ATCC® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Growth Conditions
Temperature: 37.0°C

Cost GJ, Boeke JD. A useful colony colour phenotype associated with the yeast selectable/counter-selectable marker MET15. Yeast 12: 939-941, 1996. PubMed: 8873447

Brachmann CB, et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14: 115-132, 1998. PubMed: 9483801

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