pDS472a (ATCC® 87615)

Applications: encodes removable tag for protein isolationexpression vector  /  Depositors: SL Forsburg

Designations pDS472a
Depositors SL Forsburg
Biosafety Level 1
Vector Information
Size (kb): 9.1999998092651370
Vector: pDS472a (phagemid)
Promoters: Promoter for expression nmt1 (full strength)
Construction: REP4X
Construct size (kb): 9.199999809265137
Features: marker(s): ampR
marker(s): ura4+
promoter for expression: nmt1 (full strength)
replicon: ars1
replicon: f1
replicon: pMB1
MCS: XhoI...SmaI
tag (C-terminal): bacterial GST
encodes removable tag for protein isolation
expression vector
Restriction digests of the clone give the following sizes (kb): HindIII--6.2, 1.9, 1.1; EcoRI--8.0, 1.2; PstI--9.2.
The pDS472 5' oligo changed the ATG of GST to TTG (M1L) in order to prevent translation of only the GST portion of a fusion protein.
The vector was designed for C-terminal tagging with GST (glutathione S transferase). The vector contains a thrombin cleavage site (LVPR/GS) immediately following the GST moiety, to allow removal of the tag following purification.
The vector was constructed by 1) amplification of the Schistosoma japonicum glutathione S-transferase gene with primers designed to flank the gene and modify the polylinker, 2) gel purification and digest of the PCR product with XhoI,
3) ligation into pREP4X cleaved with XhoI and SmaI; the SmaI site was lost during cloning, 4) digestion with NotI & SalI, purified GST-containing fragment was cloned into pSLF172, digested with NotI and SalI.
Media ATCC® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Growth Conditions
Temperature: 37.0°C

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

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