Plasmid Info:

Plasmid Purpose:

This vector enables the expression of transgenes in mammalian cell lines under the control of the CMV promoter. The plasmid also contains the Encephalomyocarditis virus (EMCV) Internal Ribosome Entry Site (IRES) sequence that allows for the expression of two genes from the same mRNA molecule. In this plasmid there is a cyan fluorescent protein (CFP) under the control of the IRES. The reporter gene is called Frosty CFP and was developed by DNA2.0.

IRES expression is typically significantly lower (10-30 fold less total protein by weight depending on cell type) than 5 prime cap-dependent translation (the upstream gene). For this reason detection can be difficult in cell lines that are hard to transfect. Alternative plasmids in our product range include pSF-CMV-PGK-FrCFP which typically exhibits higher expression levels.

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.The IRES that is used to drive CFP expression from the CMV promoter is considerably weaker than the CMV promoter and typically yields 15-20 fold lower protein levels.

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:

This plasmid has been designed to be compatible with a range of cloning techniques. The multiple cloning site contains a range of standard commonly used restriction sites for cloning. Using these sites genes can be inserted using standard cloning methods with DNA ligase. Other methods such as ligase independent cloning (LIC) Gibson Assembly InFusionHD or Seamless GeneArt can also be used and because all of our plasmids are based on the same backbone the same method can be used for cloning into all of our catalogue vectors.

There are a few important sites within the 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.

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