PSF-CMV-CMV-SBFI-UB-PURO - DUAL CMV EXPRESSION PLASMID

This dual expression plasmid vector contains two CMV promoters with separate MCSs together with puromycin selectable marker (driven by the ubiquitin promoter)

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.

This plasmid is a dual CMV expression vector designed for the insertion of two genes under the control of two individual CMV promoters. It enables high expression of transgenes in mammalian cells and also contains a puromycin resistance expression cassette for the creation of stable cell lines.

Multiple cloning site notes: There are a few important sites within first MCS. These include the NcoI site the XbaI site and the BsgI and BseRI sites. The NcoI site contains a start codon that is immediately downstream of both a Kozak and Shine-Dalgarno ribosomal binding site. These allow for optimal positioning of genes when the start codon is placed in this location. If this is not required and you wish to use a downstream site for gene cloning you can remove the NcoI site by cleaving the plasmid with KpnI.

The XbaI site contains a stop codon. This stop codon is positioned in a specific position in relation to the BsgI and BseRI sites that are immediately downstream. When either BseRI or BsgI cleave the plasmid they produce a TA overhang from the stop codon in the XbaI site that is compatible with all of our peptide tag plasmids cut with the same sites. BseRI and BsgI sites are non-palindromic and cleave a defined number of bases away from their binding site.

Whenever we clone a gene into our multiple cloning site we always position the start and stop codon in the same positions in the MCS. If the start and ends of the genes are not compatible with NcoI and XbaI we extend the sequence to the nearest external sites but keep the start and stop codons locations consistent.The second multiple cloning site has been designed to be compatible with the first where possible. This is achieved by in some cases using enzyme sites that produce the same overhangs as those sites in the main MCS. For example PspOMI SalI PciI AclI and SpeI produce the same overhangs as NotI XhoI NcoI ClaI and XbaI respectively. This allows gene that are located in the main MCS to be transferred to the second MCS if required. Ligating these sites together will ablate the sites from the ends of the gene sequence.

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