pRS314 plasmid in E. coli (ATCC® 77143)

Applications: YC-type (centromeric) shuttle vectorshuttle vectorvector containing primer sites useful for sequencingvector permitting RNA synthesis in vitrovector permitting production of single-stranded DNAvector permitting visual detection of recombinants  /  Depositors: P Hieter

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Designations pRS314 plasmid in E. coli
Depositors P Hieter
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): 4.785
Intact vector size: 4.785
Type of vector: phagemid
Cloning sites: KpnI ApaI XhoI SalI ClaI EcoRI PstI SmaI BamHI SpeI EagI
NotI SacII SacI NaeI
Polylinker sites: KpnI ApaI XhoI SalI ClaI HindIII EcoRV EcoRI PstI SmaI BamHI
SpeI XbaI EagI NotI BstXI SacII SacI
Construction: pRSS56 [pBluescript KS+, pBS(+)]
Host range: Saccharomyces cerevisiaeCandida robusta; Escherichia coli
Features (with orientation and position when available):
marker(s): TRP1, ->, 468-1142
replicon: f1, ->, 1463-1562
insert detection: lacZ', <-, 1696-2033
promoter for in vitro transcription: T7, ->, 1862-1881
MCS: SacI...KpnI, ->, 1889-1996
promoter for in vitro transcription: T3, <-, 2008-2027
promoter: lac, <-
replicon: pMB1, 2451-2451
marker(s): ampR, <-, 3209-4069
centromere: CEN6, 4211-4327
replicon: ARSH4, 4328-4702
Vector: pRS314 (phagemid)
Promoters: Promoter for in vitro transcription T7
Construction: pRSS56 [pBluescript KS+, pBS(+)]
Construct size (kb): 4.785
Features: insert detection: lacZ'
marker(s): TRP1
marker(s): ampR
promoter: lac
promoter for in vitro transcription: T3
promoter for in vitro transcription: T7
replicon: ARSH4
replicon: f1
replicon: pMB1
MCS: SacI...KpnI
centromere: CEN6
YC-type (centromeric) shuttle vector
shuttle vector
vector containing primer sites useful for sequencing
vector permitting RNA synthesis in vitro
vector permitting production of single-stranded DNA
vector permitting visual detection of recombinants
Restriction digests of the clone give the following sizes (kb): EcoRI--4.8; BamHI--4.8; PvuII--4.2, 0.5.
One of a series of pBluescript-based centromere vectors (ATCC 77142-77145, 77157-77158) differing in the yeast selectable marker gene.
YC-type centromere vector permitting visual detection of recombinants and production of ssDNA in E. coli. Contains promoters for in vitro RNA synthesis, priming sites useful for sequencing, and encodes the lacZ alpha (lacZ') peptide.
pRSS56, constructed by ligating a PvuI fragment (bp 498-2412) of pBluescript KS+ to a PvuI fragment (bp 2850-730) of pBS(+), contains the KS MCS from pBluescript KS+ and the unique NdeI and AatII sites between bla and f1 origin of pBS(+).
A genomic HincII/PstI fragment (1.002 kb) containing the TRP1 gene was inserted into the NdeI site and a cassette containing CEN6 and the ARS associated with histone 4 (ARSH4) was inserted into the AatII site of pRSS56. All ends were blunted.
An EcoRI site in the TRP1-containing fragment (external to the coding sequence) was destroyed.
The order of the major features in this plasmid is: TRP1 - f1 ori (NaeI) - T7 promoter - lacZ'/MCS - T3 promoter - pMB1 ori - bla - CEN6 - ARSH4.
Media ATCC® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Growth Conditions
Temperature: 37.0°C

Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27, 1989. PubMed: 2659436

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