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pUC Origin Plasmids

OG273 - pSF-CMV-COOH-EKT-His1.1


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Sequence (txt)

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-EKT-His-1.1

Product Code: OG273

Quantity Provided: 5µg

Size (bp): 4283

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 histidine purification tag that allows protein purification by binding to metal matrices such as nickel or cobalt. The His tag in this plasmid is design to be positioned at the 3’ end (C-terminus) of a gene (expressed protein) in the primary MCS. The positioning of the His tag coding sequence is such that the BsgI and BseRI restriction sites will cleave immediately upstream of the His tag coding sequence and produce a TA overhang. When any of our other plasmids that contain a gene in the main multiple cloning site (reporter genes etc) are also cut with either BsgI or BseRI these sites will also produce a TA overhang but in these plasmids the overhang will be within the stop codon of the gene. We  position all of our genes in this way in the main MCS. This allows the His tag and gene of interest to be ligated together and form a fusion protein using a single ligation step, and adding no additional nucleotides. This is possible because in this plasmid the TA overhang is followed by a C residue, creating at TAC codon, replacing the TAG stop codon that stops translation of all of the genes we insert into our main MCS. Using this system allows us to sell wild-type gene sequences that terminate correctly, but then allow you to fuse any of our fusion proteins or tags by cleaving within the gene's stop codon.  The His tag coding sequence contains six histidine residues and is positioned downstream of a small glycine spacer and also an enterokinase (EKT) cleavage site (DDDDK) that can be used to remove the tag from the purified protein if required. It cleaves after the lysine residue.

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 vector has been demonstrated to express reporter genes to high levels under the CMV promoter with His tag fused to the C-terminus of the reporter gene coding sequence. The start codon of these reporters was positioned within the NcoI site and the stop codon was positioned downstream of the His tag coding sequence. The presence of the peptide reduced the activity of the reporter gene by 2-fold, however, all of our other C-terminal tags (except GST) reduced expression by an equal amount, suggesting the effect was independent of the His 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.

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 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 distal from their recognition sites.