Description_x000D_ General description_x000D_ 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._x000D_ 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._x000D_ 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. For this reason we stock a range of other promoters that are compatible with this plasmid and are available on request._x000D_ Application_x000D_ 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:_x000D_ 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 sIt`s. There are Shine-Dalgarno sequences and KOZAK sequences aligned with the start codon of the peptide tag._x000D_ The ClaI to NheI sIt`s have other functions such as adding C-terminal peptide tags second promoters or IRES expression components. The BsgI and BseRI restriction sIt`s 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._x000D_ Sequence_x000D_ Quick-reference Plasmid Map_x000D_ Please select the file type you require. For reference most cloning programs will import a .gb (Genbank) file and will show all of the plasmids features automatically when downloaded and imported._x000D_ Genebank Vector Sequence File_x000D_ FASTA Vector Sequence File_x000D_ Full Plasmid Map_x000D_ Analysis Note_x000D_ To view the Certificate of Analysis for this product, please visit www.oxfordgenetics.com._x000D_ Other Notes_x000D_ Looking for more vector options to move your experiments forward faster? Consider a custom cloning vector designed and built by Oxford Genetics™. Find out more at Oxford Genetics - Sigma's partner for cloning and expression vectors for molecular biology and synthetic biology applications._x000D_ Legal Information_x000D_ Oxford Genetics is a trademark of Oxford Genetics Ltd