pSLF172 (ATCC® 87609)

Applications: encodes an epitope tag for protein isolation or monitoringexpression vector  /  Depositors: SL Forsburg

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Designations pSLF172
Depositors SL Forsburg
Biosafety Level 1
Host
Distribution host: Escherichia coli HB101 (ATCC 33694)
Vector Information
Size (kb): 8.5000000000000000
DESCRIPTION OF VECTOR:
Intact vector size: 8.500
Type of vector: phagemid
Cloning sites: XhoI BglII NotI BamHI SalI SmaI
Polylinker sites: XhoI BglII NotI HA triple tag BamHI TAG SalI SmaI
Construction: REP4X
Host range: Schizosaccharomyces pombe; Escherichia coli
Features (with orientation and position when available):
marker(s): ampR
marker(s): ura4+
replicon: ars1
replicon: pMB1, f1
promoter for expression: nmt1 (full strength), ->
MCS: XhoI...SmaI, ->
epitope tag: hemagglutinin (HA) triple tag
Vector: pSLF172 (phagemid)
Promoters: Promoter for expression nmt1 (full strength)
Construction: REP4X
Marker(s):ampR,ura4+
Construct size (kb): 8.5
Features: marker(s): ampR
marker(s): ura4+
promoter for expression: nmt1 (full strength)
replicon: ars1
replicon: pMB1, f1
MCS: XhoI...SmaI
epitope tag: hemagglutinin (HA) triple tag
Applications
encodes an epitope tag for protein isolation or monitoring
expression vector
Comments
Restriction digests of the clone give the following sizes (kb): HindIII--5.8, 1.7, 1.0; EcoRI--7.4, 1.1; BglII--8.5.
The fission yeast tagging vectors, pSLF172 (ATCC 87609), pSLF272 (ATCC 87610) and pSLF372 (ATCC 87611), contain three versions of the nmt1 promoter: full strength (nmt1), medium strength (nmt1*) and low strength (nmt1**), respectively.
The weaker promoters (nmt1* and nmt1**) contain mutations that attenuate both repressed and induced levels of expression.
Each version of the nmt1 promoter can be expressed at low or high levels in thiamine-free media.
The vector was designed to tag expressed protein at C-terminus with triple HA tag, which contains an internal BamHI site. The vector does not contain an ATG, which must be provided by the insert.
The vector was constructed by 1) amplification by PCR with primers designed to flank the triple HA tag in Bluescript-HA and to modify the polylinker, 2) gel purification of the PCR product and digest with XhoI
and 3) ligation into REP4X cleaved with XhoI and SmaI.
Media Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Growth Conditions
Temperature: 37.0°C
References

Forsburg SL, Sherman DA. General purpose tagging vectors for fission yeast. Gene 191: 191-195, 1997. PubMed: 9218719

Basi G, et al. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123: 131-131, 1993. PubMed: 8422997

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