Product Name: pSF-CAG-Amp
Product Code: OG504
Size (bp): 5431 bp
Bacterial Antibiotic Selection: AmpR
Origin and Compatibility: pUC high copy derived from pBR322
Bacterial Copy Number: 500-700 per cell
Promoter: CAG synthetic mammalian promoter and Ubiquitin promoter / Chicken Beta Actin (CBA) promoter
This mammalian expression vector contains the chimeric CAG promoter which is a composite of the CMV enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron. This promoter is often used instead of the CMV promoter because in some cell types the CMV promoter can get silenced. To prevent this the CAG promoter contains the CpG island from the CBA promoter to help prevent promoter methylation. The plasmid can be used to drive protein expression in a range of mammalian cell types and we have consistently found that expression levels are equal to CMV in short term experiments in standard cell types.Promoter Expression Level:
This plasmid contains the mammalian CAG promoter which is a synthetic composite of the CMV immediate early enhancer followed by the CBA promoter and the rabbit beta globin intron. The Chicken beta actin contains a CpG island that can help to keep the promoter active for longer in stable culture when compared to the CMV promoter.
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 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.Multiple cloning site notes:
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.