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GST Tag Plasmids

OG310 - pSF-CMV-COOH-EKT-GST1

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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

Product Code: OG310

Quantity Provided: 5µg

Size (bp): 4889

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 because of the stop codon in the site). The GST tag coding sequence is 219 amino acids in length. There is an enterokinase cleavage site  immediately upstream of this tag (DDDDK) that can be used to remove the tag from a purified protein, it  cleaves after the lysine residue. 
       
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 sequences and details of these primers are available through our website.

Intellectual Property Status: According to our IP-friendly policy, this plasmid is sold free of reach-through rights on any derivatives you may create.

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.