Plasmid Info:

Plasmid Purpose:

A versatile expression vector for the production of two proteins under the control of two separate CMV promoter expression cassettes in mammalian cells. This vector also contains a Kanamycin resistance cassette for growth and maintenance in E.coli.

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 in the NcoI site that can be removed by digestion with KpnI if required. The first MCS for gene insertions extends from NotI to XbaI but the Shine-Dalgarno sequences and Kozak sequences are aligned with the start codon in the NcoI restriction site. The ClaI to NheI sites have other functions such as adding peptide tags or 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.

The second MCS in this plasmid extends from PspOMI to SpeI. This MCS contains a Kozak sequence immediately upstream of the start codon in the PciI restriction site. We insert the start codons of our genes into here where possible.

This MCS has been designed to be compatible with genes that are within our main MCS by using enzymes sites in the same order that produce compatible cohesive ends that can be ligated together. This allows genes to be transferred from the main MCS (NotI to NheI) into the second MCS (PspOMI to SpeI) is required. Enzymes that are compatible between these two MCSs include:

• PspOMI compatible with NotI
• ScaI compatible with EcoRV (Blunt)
• SalI compatible with XhoI
• PciI compatible with NcoI and BspHI
• AclI compatible with ClaI
• SpeI compatible with XbaI and NheI Avr2

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