Plasmid Info:

Plasmid Purpose:

This vector contains the internal ribosome entry site (IRES) from Encephalomyocarditis virus (EMCV) between the promoter and the multiple cloning site (MCS). It is designed to be used to express genes using cap-independent expression systems although the mRNAs produced from this vector contain a 5 prime cap. This vector is not designed for the expression of two proteins from the same mRNA. For this purpose we have designed pSF-CMV-EMCV-PciI where the PciI site contains start codons in the correct position for the IRES to express efficiently. In this vector there is an NcoI site (which contains an ATG start codon) in the correct position to enable efficient expression of a gene in the MCS.

We have also developed this same vector with the T7 promoter upstream of the IRES to allow the expression of genes using T7 eukaryotic systems.

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 start codon within the NcoI restriction site that is positioned to allow efficient expression from the EMCV IRES. The main MCS for gene insertions in this vector therefore extends from the NcoI restriction site to the XbaI restriction site. The downstream sites (ClaI to NheI sites) have alternate functions and are used to insert other DNA sequences that we sell however they can be used to insert a gene if these functions are not required. These functions include adding peptide tags or downstream IRES elements. The BsgI and BseRI restriction sites cleave within the stop codon in the XbaI site and allow the retrospective fusion of coding sequences. These sites are normally only used on genes that we sell in the main multiple cloning site.

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