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Mammalian Selection Plasmids

OG87 - pSF-CMV-NH2-InsulinSP2

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Full Plasmid Details (PDF)

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.

Product Code: OG87

Size (bp): 4330     

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 the secretory signal peptide that normally induces the secretion of insulin in humans. It will traffic any protein to which it is attached at the N-terminus into the endoplasmic reticulum. It is positioned upstream of the multiple cloning site. If your protein has no organelle retention signals, or specific trafficking sequences, it will most likely be secreted into the medium by bulk flow exocytosis mechanisms.

This tag will translocate the protein to which it is attached into the endoplasmic reticulum (ER) of a eukaryotic cell, after which the signal peptide will be cleaved off. The signals that traffic the protein to the ER are within the tag, as are the signals that mediate its cleavage from the downstream protein. Its activity is based more on the hydropathy of the signal peptide sequence, rather than the actual order of the amino acids within it. The amino acid sequence of the signal peptide is MALWMRLLPLLALLALWGPDPAAA. Cleavage occurs immediately after the final alanine 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 codons are two bases out of frame with the ATG start codon that is within the NcoI restriction site.

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, and secrete, reporter genes to high levels under the CMV promoter, with the insulin signal peptide at the N-terminus of the protein directing ER translocation. The start codon of these reporters was positioned at the start of the Insulin secretory 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.
  • Human insulin signal peptide – The coding sequence of this insert has been modified to remove a BamHI site. It conserves the amino acid sequence of the wild-type protein.

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.