pGFLEX plasmid in E. coli
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)
Size (kb): 6.143
Intact vector size: 6.143
Type of vector: phagemid
Cloning sites: ClaI NotI ApI
Polylinker sites: BamHI EcoRI SmaI SalI XhoI NotI ApaI
Other unique sites: BglII BssHI EcoRV PstI PvuI ScaI SpeI
Construction: pUC19, pGPi-2, pIC-20H
Host range: mammalian cells; Escherichia coli
Features (with orientation and position when available):
Promoter: CMV, ->, 1-686
Promoter: lac, ->, 687-847
Leader Peptide: beta-gal leader sequence, 848-886
Coding Sequence: human GST pi, ->, 887-1516
Other: thrombin cleavage site, 1517-1524
MCS: BamHI...ApaI, 1525-1566
Stop Codons: TGA, 1570-1572
Replicon: f1 ori, 1895-2413
Promoter for Expression: SV40, ->, 2466-2800
Replicon: SV40 ori, 2669-2754
Coding Sequence: Neo, ->, 2836-3630
Terminator: SV40 polyadenylation, 3804-3939
Replicon: pMB1, 4373-4373
Marker(s): ampR, <-, 5131-5991
Encodes removable tag for protein isolation
Vector permitting construction of fusion proteins
Vector permitting production of single-stranded DNA
Restriction digests of the clone give the following sizes (kb): PstI--6.2; XhoI--3.7, 2.0,0.3; SalI--2.4, 2.1, 1.6.
The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST pi protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin.
High level of expression in E. coli, cos and other mammalian cultured cell lines.
Recommendation for verification: StuI--4.84, 1.30; SalI--2.4, 2.18, 1.56; PstI--6.14; NotI--6.14.
Manoharan HT, et al. High-level production and purification of biologically active proteins from bacterial and mammanlian cells using the tandem pGFLEX expression system. Gene 193: 229-237, 1997. PubMed: 9256081
Herbert Manoharan, personal communication