Plasmid Info:

Plasmid Purpose:

This vector adds a His tag that allows protein purification by binding to metal matrices such as nickel or cobalt or antibodies raised against the His tag. 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 but XbaI is ablated in this vector). The His tag coding sequence contains six histidine residues that is downstream of a small 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.

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.

Sequence and Map:

Other Info:

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.

Cloning:

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.

IP Status:

This product is part of our SnapFast plasmid range, for more information on the Intellectual property status of this plasmid please click here