Plasmid Info:

Plasmid Information

Product Name: pSF-EF1-Alpha-Cas9WT

Product Code: OG3539

Size (bp): 8977 bp

Bacterial Antibiotic Selection: KanR

Origin and Compatibility: pUC high copy derived from pBR322

Bacterial Copy Number: 500-700 per cell

Promoter: Human Elongation Factor Alpha Promoter

Plasmid Purpose:

This plasmid is designed to express wild type Cas9 in mammalian cells. Expression is driven from a EF1-Alpha promoter in the main MCS.

The EF1 Alpha promoter is a relatively strong mammalian promoter showing good activity in most mammalian cell lines. Although it exhibits weaker expression levels compared to promoters such as CMV or CAG, it is reported to maintain expression levels for longer in extended culture or in vivo. This is in part due to the large intron within the promoter.

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.

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