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OGS602

Sigma-Aldrich

PSF-CMV-UB-PURO ASCI-EMCV-FLUC - EMCV IRES LUCIFERASE PUROMYCIN SELECTION VECTOR

plasmid vector for molecular cloning

Synonym(s):
cloning vector, expression vector, molecular cloning vector, plasmid, plasmid vector, snapfast vector, vector

form

buffered aqueous solution

mol wt

size 8368 bp

bacteria selection

kanamycin

mammalian cells selection

puromycin

Origin of replication

pUC (500 copies)

Peptide cleavage

no cleavage

Promoter

Promoter name: CMV
Promoter activity: constitutive
Promoter type: mammalian

reporter gene

firefly luciferase

shipped in

ambient

storage temp.

−20°C

General description

This vector contains the CMV promoter upstream of the internal ribosoms entry site (IRES) from Encephalomyocarditis virus (EMCV) which is driving the expression of the firefly luciferase reporter gene. The vector also contains a puromycin selection cassette that allows the production of stable cell lines in mammalian cells. Transgenes can be inserted upstream of teh EMCV IRES sequence using our standard multiple cloning site. This will allow both the gene of interest and the luciferase to be produced from the same mRNA molecule.

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. The IRES that is used to drive luciferase expression from the CMV promoter is considerably weaker than the CMV promoter and typically yields 15-20 fold lower protein levels.

Application

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

To view sequence information for this product, please visit the product page

Analysis Note

To view the Certificate of Analysis for this product, please visit www.oxgene.com

Storage Class Code

12 - Non Combustible Liquids

Flash Point(C)

Not applicable

Certificate of Analysis

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Certificate of Origin

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