Product Name: pSF-CMV-Gag-Pol (SnapFast Pro™)
Product Code: OG637
Size (bp): 8607 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
This plasmid encodes a codon optimised version of the Human Immunodeficiency Virus (HIV) Gag-Pol protein gene under transcriptional regulation of the strong mammalian CMV promoter. In the life cycle of HIV the Gag protein is expressed in high amounts, however, every 1:20 times during translation of the Gag coding sequence the ribosome shifts backwards by one base. The ribosome then continues to translate the protein as a Gag-Pol fusion. The region of frameshift has not been optimised in this plasmid to ensure that this process continues. It is important to note that the Gag-Pol fusion protein is required for virion maturation and as such the Gag cassette cannot be separated from the Pol cassette because Pol protein alone will not be incorporated into the assembling virion. This is because Gag localises to the lumenal side of the cell membrane during virus budding.
Gag-Pol is proteolytically cleaved to produce a number of smaller proteins. The protease itself is self-cleaving and derived from the Pol protein section. The protease is toxic to mammalian cells.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.
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. 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:
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.
This product is part of our SnapFast™ plasmid range, for more information on the intellectual property status of this plasmid and the terms of our licences please click here.