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OG390 - pSF-CMV-PGK-daGFP

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This plasmid is compatible with all of the other DNA sequences available on this site, which are all sold in the same backbone. This flexibility allows you to create any vector you require, with a variety of functions, simply by compiling the relevant sections from our product range.

Vector: pSF-CMV-PGK-daGFP

Product Code: OG390

Quantity Provided: 5µg

Size (bp): 5452

Bacterial Antibiotic Selection: Kanamycin

Origin and Compatibility: pUC high copy, derived from pBR322 

Copy Number: 500-700 copies per cell

Promoter:  Cytomegalovirus (CMV) immediate early promoter and the mouse phosphoglycerate kinase promoter (PGK)

Purpose:  This plasmid contains two promoters that terminate transcription at the same poly-adenylation signal, allowing the expression of two genes from one expression cassette, where the second gene is the  daGFP fluorescent reporter gene. The second promoter (PGK) is approximately 10-fold weaker than the upstream CMV promoter in most commonly used cell lines.

daGFP: This vector encodes a green fluorescent protein called daGFP. This protein can be used in the same way as normal GFP using argon laser based or UV based excitation apparatus to allow the detection of fluorescence. The protein has a peak excitation of 510nm and a peak emission of 521nm. The gene encoding the protein is sold free of intellectual property restrictions.  

Quality Validation: This vector has been demonstrated to express reporter genes to high levels under the CMV promoter. The start codon of these reporters was positioned within the NcoI site and the stop codon was positioned within the XbaI site. This plasmid also demonstrates intermediate levels of expression of daGFP from the PGK promoter. Expression has been validated in 293 cells.

Quality Sequencing Analysis: This vector has been fully sequenced using primers F1-F10. The sequences and details of these primers are available through our website.

Intellectual Property Status: According to our IP-friendly policy, this plasmid is sold free of reach-through rights on any derivatives you may create.

Genetic Modifications to standard parts:

  • CMV promoter – an NcoI site has been ablated in the CMV promoter. This change does not reduce expression.
  • KanR cassette – This KanR resistance region is not a wild type sequence, it has been modified to remove all restriction sites that conflict with the SnapFast system and to retain a high level of antibiotic resistance. It has been validated in E.coli.
  • daGFP – The daGFP reporter gene has been modified to remove PciI and BseRI restriction sites.

Restriction site notes:

  • Bgl2, KpnI, SwaI and PmeI each cut the vector at two sites positioned to flank the promoter, start codon, origin, and KanR, respectively.
  • BsgI and BseRI recognise non-palindromic DNA sequences and cleave upstream of their recognition sites, within the stop codon that is found inside the XbaI restriction site.