Adds a C terminal His tag (frame 3) to a gene inserted into our main multiple cloning site. Also contains the CMV promoter to drive transcription.
Product Name: pSF-CMV-COOH-EKT-His3
Product Code: OG68
Size (bp): 4270 bp
Bacterial Antibiotic Selection: KanR
Origin and Compatibility: pUC high copy derived from pBR322
Bacterial Copy Number: 500-700 per cell
Promoter: Cytomegalovirus (CMV) immediate early promoter
This vector adds a His tag that allows protein purification by binding to metal matrices such as nickel. The His tag will be positioned at the 3 prime end (C terminus) of a gene (expressed protein) in the primary MCS (NotI to XbaI). The His tag coding sequence contains six histidine residues that is downstream of a short glycine spacer and an enterokinase cleavage site (DDDDK) that can be used to remove the tag from a purified protein if required. It cleaves after the lysine residue.
Frame: For reference the coding sequence of the HIS tag is one base pair out of frame with the TAG stop codon that is found within the upstream XbaI site.Promoter Expression Level:
This plasmid contains the mammalian CMV promoter to drive gene expression. We have tested all of our mammalian promoters in a range of cell types and CMV is consistently the strongest in those we have studied. However there are many reports of the CMV promoter demonstrating silencing by methylation in long-term culture. For this reason we stock a range of other promoters that are compatible with this plasmid and are available on request.
This plasmid contains three alternative transcription terminators for mammalian bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.
There is a start codon in the NcoI site can be removed by digestion with KpnI if required. The MCS for gene insertions extends from NotI to XbaI. There are built in Shine-Dalgarno and KOZAK sequences aligned with the start codon within the NcoI restriction site. If these are removed you will need to add a ribosomal entry site to the 5 prime end of your gene for optimal expression.