pPW264 (ATCC® 87443)

Applications: vector permitting positive selection for inserts  /  Depositors: F Puta

Designations pPW264
Permits and Restrictions

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Depositors F Puta
Biosafety Level 1
Vector Information
Size (kb): 8.1070003509521480
Vector: pPW264 (plasmid)
Promoters: Promoter lambda PR
Construction: pML25, pPW263
Construct size (kb): 8.107000350952148
Features: marker(s): URA3
marker(s): ampR
marker(s): cmlR
operator: lac
promoter: lambda PR
promoter: tetR
replicon: 2 micron
replicon: pMB1
repressor gene: cI
transcription terminator: tandem fd terminators
vector permitting positive selection for inserts
Restriction digests of the clone give the following sizes (kb): HindIII--8.2; EcoRI--7.2, 0.96; BglII--8.2.
The cytoplasmic chloramphenicol resistance protein does not negatively affect cell growth when overproduced (in contrast to cloning vectors that overexpress membrane associated tetR protein).
The level of resistance to chloramphenicol differs substantially depending on the stage at which they are tested.
The chloramphenicol resistance of the plasmid should be tested before cloning inserts.
YE-type shuttle vector allowing positive selection of inserts by conferring chloramphenicol resistance (recommended concentration 10 ug/ml) when a sequence has been inserted into the cI repressor gene.
A pair of phage fd rho-independent strong terminators downstream of the cmlR gene prevents transcription through the origin of replication. This stabilizes recombinant plasmid copy number when the PR promoter for the cmlR gene is derepressed.
Media ATCC® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Growth Conditions
Temperature: 37.0°C

Puta F, Wambutt R. Construction of a new Escherichia coli-Saccharomyces cerevisiae shuttle plasmid cloning vector allowing positive selection for cloned fragments. Folia Microbiol. 37: 193-198, 1992. PubMed: 1505881

Puta F, et al. Direct selection shuttle plasmid vector, pPW264, used for cloning the alpha-amylase gene of Schwanniomyces occidentalis. Folia Microbiol. 39: 255-260, 1994. PubMed: 7729761

Frantisek Puta, personal communication

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