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Flag Tag Plasmids

OG308 - pSF-CMV-COOH-FLAG1

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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-COOH-FLAG1

Product Code: OG308

Quantity Provided: 5µg

Size (bp): 4241

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

Purpose: This vector adds a FLAG tag that allows protein detection or purification using antibodies against the peptide. The FLAG tag will be positioned at the 3’ end (C-terminus) of a gene (expressed protein) in the primary MCS (NotI-XbaI, but XbaI is ablated in this vector because of the stop codon in the site). The FLAG tag coding sequence is DYKDDDDK. This sequence contains an enterokinase cleavage sequence (DDDDK), however, when used as a C-terminal tag this will not remove the tag because it cleaves after the final lysine, which will be the final amino acid in any protein produced from this vector. The FLAG tag coding sequence is positioned immediately downstream on the XhoI restriction site.      

Quality Validation: This vector has been demonstrated to express reporter genes to high levels under the CMV promoter with a FLAG tag fused to the C-terminus of the reporter gene. The start codon of these reporters was positioned within the NcoI site and the stop codon was positioned at the end of the FLAG tag. The presence of the peptide reduced the activity of the reporter gene by 2.1-fold, however, all of our other C-terminal tags (except GST) reduced expression by an equal amount, suggesting the effect was independent of the FLAG tag sequence itself, and only an inherent property of the reporter used. Expression was validated in the A549 lung carcinoma cell line.

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.

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.