Analysis Note

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Application

Cloning in a gene: PSF-LENTI - lentivirus puromycin CMV 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.

General description

PSF-LENTI - lentivirus puromycin CMV plasmid contains a HIV1 based lentivirus expression vector that can be used for the stable inteagration of genes into both dividing and non-dividing cells. The plasmid is based on the commonly used pCCL lentivirus backbone and contains a range of modifications to make cloning easier. The plasmid vector contains an expanded multiple cloning site and is compatible with most of the plasmids we provide in our product range which allows sections to compiled to create complex expression vectors. This plasmid contains the CMV promoter to drive the expression of a gene of interest followed by a multiple cloning site and the PGK promoter to drive puromycin resistance. 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. The murine phosphoglycerate kinase (PGK) promoter driving puromycin expression allows for long term stable expression in vitro and in vivo in most types.

Sequence

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