Product Name: pSF-CMV-FLuc-UB-Cas9WT-BgH_Pac1 (SnapFast Pro™)
Product Code: OG3529
Size (bp): 11548 bp
Bacterial Antibiotic Selection: AmpR
Origin and Compatibility: pUC high copy derived from pBR322
Bacterial Copy Number: 500-700 per cell
Promoter: Cytomegalovirus (CMV) immediate early promoter / Human Ubiquitin Promoter
This plasmid is designed for expressing wild-type Streptococcus pyogenes Cas9 protein in mammalian cells. Cas9 is the DNA endonuclease central to the CRISPR genome editing system. Expresssion of Cas9 is driven by the ubiquitin promoter. The plasmid also contains a firefly luciferase reporter gene whose expression is controlled by a CMV promoter in the main MCS. For more information on CRISPR gene editing read our guide to CRISPR technologies or view a complete list of our CRISPR plasmids.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. For this reason we stock a range of other promoters that are compatible with this plasmid and are available on request.
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. The Cas9 protein is terminated by the Bovine growth hormone polyA signal.
This plasmid contains a gene (FLuc)within the main multiple cloning site (NotI-ClaI). Any plasmid that we sell where the gene is this configuration will be located in the exact same position in relation to the start and stop codon of the gene in the plasmid. The only exceptions to this rule are fusions proteins where the fusion gene may be positioned at the front or end of the MCS to allow gene fusion.
By positioning all of our genes in the same location it allows them to be transferred between plasmids using the same cloning method and restriction sites regardless of the plasmid being used from our product range. Inserting a new gene into this plasmid should be easily possible using a range of standard restriction enzyme sites that flank the gene currently in the vector.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.
BseRI and BsgI sites are non-palindromic and cleave a defined number of bases away from their binding sites. This allows them to cut the upstream stop codon in the gene in this plasmid regardless of the gene sequence.