Plasmid Info:

Plasmid Purpose:

Plasmid vector for expressing wild-type Streptococcus pyogenes Cas9 protein in mammalin cells from a CMV enhancer promoter expression cassette in the main pSF MCS. Also contains a down-stream Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) for the co-expression of the puromycin resistance gene.

This plasmid contains the enhancer region from the CMV promoter with a multiple cloning site downstream. This allows the insertion of promoters downstream of the enhancer which can be used as a way to increase the expression of weaker promoters. This plasmid will show some basal gene expression in the absence of a promoter inserted because the CMV enhancer region alone can lead to some transcription initiation.

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 contains a gene 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.

This plasmid also contains an internal ribosome entry site (IRES) followed by a puromycin selection gene (Puro). The IRES allows the co-expression of two genes from the same mRNA, in this case both Cas9 and Puro. When designing all our plasmids we aim to remove all conflicting resitriction sites to make cloning simpler, however, the IRES contains a number of restriction sites that are within coformationally important hairpins, we have therefore been unable to remove them. therefore, please check this plasmid map before cloning to ensure none of these restriction sites will cause cloning problems.

In the multiple cloning site there are two important restriction sites called BsgI and BseRI sites. These sites both cut the DNA at the same position and cleave the stop codon of the gene in the multiple cloning site in this plasmid thereby producing a TA overhang. This overhang is compatible with any of our peptide or reporter fusion tag plasmids also cut with either of these enzymes. This allows seamless C-terminal fusions to be made with the gene in this multiple cloning site using a single cloning step from our C-terminal peptide and reporter tag product range. Normally the easiest method is to clone the C-terminal tag from our other plasmid products into this plasmid using BsgI or BseRI and the downstream ClaI restriction site.

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.

IP Status:

This product is part of our SnapFast Pro plasmid range, for more information on the Intellectual property status of this plasmid please click here