Analysis Note

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Application

Cloning in a gene: This vector has been designed to allow the addition of a peptide tag to the end of a protein of interest using standard cloning techniques.Multiple Cloning Site Notes: There is a start codon in the NcoI site can be removed by digestion with KpnI if required. The MCS for gene insertions extends from NotI to XbaI however the tag resides between the NotI and HindIII sites. There are Shine-Dalgarno sequences and KOZAK sequences aligned with the start codon of the peptide tag.The ClaI to NheI sites have other functions such as adding C-terminal peptide tags second promoters or IRES expression components. The BsgI and BseRI restriction sites cleave within the stop codon in the XbaI site and allow the retrospective fusion of C-terminal peptide tags sequences if the stop codon is placed in this position.

General description

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 but adjacent to the NcoI restriction 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. Promoter Expression Level: This plasmid contains the mammalian CMV promoter to drive gene expression. We have tested all of our mammalian promoters in a range of cell types and CMV is consistently the strongest in those we have studied. However there are many reports of the CMV promoter demonstrating silencing by methylation in long-term culture.

Sequence

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