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Flag Tag Plasmids

OG96 - pSF-CMV-NH2-HA-FLAG-EKT-Ncol

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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.

Product Code: OG96

Size (bp): 4247   

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 plasmid adds a FLAG epitope tag to the N-terminus of a protein that is encoded within the multiple cloning site. This tag allows the detection and purification of a tagged protein using antibodies raised against the FLAG epitope. The FLAG tag coding sequence is DYKDDDDK. There is an enterokinase cleavage site (DDDDK) within the FLAG tag sequence that can be used to remove it from a purified protein if required. It cleaves after the lysine residue.

Transcription Termination: This plasmid contains three alternative transcription terminators for mammalian, bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.

Frame: The tag is in frame with the ATG start codon that is within the NcoI restriction site, allowing fusion with any genes that we sell in the MCS that contain an NcoI at the 5’. This tag is positioned immediately upstream of the NcoI site to minimise the amount of extra amino acids that are added to the N-terminus. We also sell the same tag positioned further upstream, flanked by NotI and HindIII (pSF-CMV-NH2-FLAG- EKT1) and in two alternative frames (pSF-CMV-NH2-FLAG-EKT2 and 3).

Inserting a Gene: Each restriction site in this vector has a purpose and allows the insertion of specific pre-designed DNA sequences. To insert a gene, we recommend using any sites between (and including) the HindIII and XbaI sites. By doing this you will be able to insert most of our other products upstream, or downstream, of your gene. Click here to read more about the purpose of each restriction site.

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

Quality Validation: This plasmid has been demonstrated to express reporter genes to high levels under the CMV promoter, with the FLAG peptide tag at the N-terminus of the protein. The start codon of these reporters was positioned at the start of the C-Myc tag and the stop codon was positioned within the XbaI restriction site. Reporter gene 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.

 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 SnapFastTM 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 this plasmid 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.