N-terminal His epitope expression plasmid (Frame 2). Adds a Hexa-His tag to the start of your gene of interest and also has an EKT cleavage tag.
Product Name: pSF-CMV-NH2-His-EKT2
Product Code: OG331
Size (bp): 4290 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 to the N-terminus of a protein that is encoded within the multiple cloning site. The His tag coding sequence contains six histidine residues that is upstream of 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. The His tag allows the purification of a tagged protein by binding to metal matrices such as nickel or cobalt. The tag can also be used to allow the detection of tagged proteins using antibodies raised against the His tag.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.
This vector has been designed to allow the addition of a peptide tag to the end of a protein of interest using standard cloning techniques.Multiple Cloning Site Notes:
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 however the tag resides between the NotI and HindIII sites. There are Shine-Dalgarno sequences and KOZAK sequences aligned with the start codon of the peptide tag.
The ClaI to NheI sites have other functions such as adding C-terminal peptide tags second promoters or IRES expression components. The BsgI and BseRI restriction sites cleave within the stop codon in the XbaI site and allow the retrospective fusion of C-terminal peptide tags sequences if the stop codon is placed in this position.