pRS402 plasmid in E. coli (ATCC® 87477)

Applications: YI-type (integrating) shuttle vectorvector permitting visual detection of recombinants phosphoribosylaminoimidazole succinocarboxamide synthetase SAICAR synthetase, phosphoribosylaminoimidazole carboxylase  /  Depositors: JD Boeke

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Designations pRS402 plasmid in E. coli
Depositors JD Boeke
Biosafety Level 1
Host
Distribution host: Escherichia coli HB101 (ATCC 33694)
Vector Information
Size (kb): 5.528
DESCRIPTION OF VECTOR:
Intact vector size: 5.528
Type of vector: phagemid
Cloning sites: SacI SacII EagI NotI SpeI BamHI SmaI PstI EcoRI 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, pASZ11
Host range: Saccharomyces cerevisiae; Escherichia coli
Features (with orientation and position when available):
marker(s): ADE2, ->, 575-2290
replicon: f1, <-, 2495-2951
promoter for in vitro transcription: T7, ->, 3121-3140
MCS: KpnI...SacI, ->, 3148-3250
promoter for in vitro transcription: T3, <-, 3267-3286
insert detection: lacZ', <-, 2952-3311
promoter: lac, <-, 3356-3384
replicon: pMB1, 3710-3710
marker(s): ampR, <-, 4468-5328
Vector: pRS402 (phagemid)
Promoters: Promoter for in vitro transcription T7
Construction: pJK142, pASZ11
Marker(s):ampR,ADE2
Construct size (kb): 5.528
Features: insert detection: lacZ'
marker(s): ADE2
marker(s): ampR
promoter: lac
promoter for in vitro transcription: T3
promoter for in vitro transcription: T7
replicon: f1
replicon: pMB1
MCS: KpnI...SacI
Applications
YI-type (integrating) shuttle vector
vector permitting visual detection of recombinants phosphoribosylaminoimidazole succinocarboxamide synthetase SAICAR synthetase, phosphoribosylaminoimidazole carboxylase
Comments
Restriction digests of the clone give the following sizes (kb): BamHI--5.5; BglII--3.3, 2.2; EcoRI--5.5.
ade2 phenotype produces red colonies when grown on adenine 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 ADE2 gene from pRS402, 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 a MET15 marker gene disruption product from pRS401 (ATCC 87473).
pRS402 can be used to generate a gene specific ADE2 marker gene disruption cassette for tranformation in gene knockout experiments.
The 20nt PCR primer sequences for generating the ADE2 marker from pRS402 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
References

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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Shipped frozen
Shipping Information Distributed: freeze-dried
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