PSF-CMV-UB-FRCFP - MAMMALIAN CFP REPORTER VECTOR OGS512-5UG

NACRES:

NA.85

A CMV expression plasmid that allows for the insertion of exogenous transgenes for expression in mammalian cells. The plasmid also contains a reporter gene cassette away from the multiple cloning site that allows for high levels of expression of Cyan Fluorescent protein. The reporter in this plasmid was original developed by DNA2. 0 and is called Frosty CFP.
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 human ubiquitin promoter driving reporter gene expression is the strongest mammalian endogenous promoters that we provide for many cell types.
Cloning in a gene: This 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.
Sequence
Analysis Note

form	buffered aqueous solution
mol wt	size 6263 bp
Origin of replication	pUC (500 copies)
Peptide cleavage	no cleavage
Promoter	Promoter name: CMV Promoter activity: constitutive Promoter type: mammalian
bacteria selection	kanamycin
reporter gene	CFP
shipped in	ambient
storage temp.	20°C

RIDADR	NONH for all modes of transport
Flash Point F	Not applicable
Flash Point C	Not applicable