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GST Tag Plasmids

OG274 - pSF-CMV-COOH-EKT-GST1.1

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Full Plasmid Details (PDF)

Sequence (txt)

This plasmid is compatible with all of the other DNA sequences available on this site, which are all sold in the same backbone. This flexibility allows you to create any vector you require, with a variety of functions, simply by compiling the relevant sections from our product range.

Vector: pSF-CMV-COOH-EKT-GST1.1 

Product Code: OG274

Quantity Provided: 5µg

Size (bp): 4913

Bacterial Antibiotic Selection: Kanamycin

Origin and Compatibility: pUC high copy, derived from pBR322   

Copy Number: 500-700 copies per cell

Promoter: Cytomegalovirus (CMV) immediate early promoter

Purpose: This vector adds a Glutathione-S-transferase (GST) purification tag that allows proteins to be purified by binding to glutathione. The GST tag will be positioned at the 3’ end (C-terminus) of a gene (expressed protein) in the primary MCS (NotI-XbaI, but XbaI is ablated in this vector). The positioning of the GST tag coding sequence is such that the BsgI and BseRI restriction sites will cleave immediately upstream of the GST tag coding sequence and produce a TA overhang. When any of our other plasmids that contain a  gene in the main multiple cloning site (reporter genes etc) are also cut with either BsgI or BseRI these sites will also produce TA overhang but in these plasmids the overhang will be within the stop codon of the gene. We position all of our genes in this way in the main MCS. This allows the GST tag and gene of interest to be ligated together and form a fusion protein using a single ligation step, and adding no additional nucleotides. This is possible because in this plasmid the TA overhang is followed by a C residue, creating at TAC codon, replacing the TAG stop codon that stops translation of all of the genes we insert into our main MCS. Using this system allows us to sell wild-type gene sequences that are terminated correctly, but then allow you to fuse any of our fusion proteins or tags by cleaving within the genes stop codon.

Quality Validation: This vector has been demonstrated to express reporter genes to high levels under the CMV promoter with a GST tag fused to the C-terminus of the reporter gene. The presence of the GST tag reduced the activity of the reporter gene by approximately 5-fold, although this appeared to be due to the length of the tag rather than its sequence, because other tags of similar sizes cause an equal reduction. The start codon of these reporters was positioned within the NcoI site and the stop codon was positioned at the end of the GST tag. Expression was validated in the A549 lung carcinoma cell line.   
Quality Sequencing Analysis: This vector has been fully sequenced using primers F1-F10. The details and sequences of these primers are available through our website.

Genetic Modifications to standard parts: 
•    CMV promoter – an NcoI site has been ablated in the CMV promoter. This change does not reduce expression. 
•    KanR cassette – This KanR resistance region is not a wild type sequence, it has been modified to remove all restriction sites that conflict with the SnapFast system and to retain a high level of antibiotic resistance. It has been validated in E.coli.
•    The GST tag has been optimised to remove all restriction sites that conflict with the SnapFastTM system. These changes conserve the amino acid sequence of the original protein. 

Restriction site notes: 
•    Bgl2, KpnI, SwaI and PmeI each cut the vector at two sites positioned to flank the promoter, start codon, origin, and KanR, respectively. 
•    BsgI and BseRI recognise non-palindromic DNA sequences and cleave distal from their recognition sites.