This plasmid is designed to express tagged proteins in mammalian cells either by transient transfection or by creating stable cell lines. It contains a puromycin resistance expression cassette using the human Ubiquitin promoter to drive expression and allow for the selection of cells containing the plasmid.
About the Peptide Tag:This plasmid contains an n-terminal Maltose Binding Protein (MBP) affinity tag that can be fused to a gene of interest to allow protein detection and/or purification. The sequence of the tag is: EEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSS.
About the Cleavage Tag:This plasmid also encodes a protease cleavage site that is designed to be positioned between your gene of interest and the tag to allow the removal of the tag following protein purification or isolation. This plasmid contains a FXa cleavage tag. The protein sequence of the cleavage tag is: IEGR. It cleaves after the Arg residue but can also cleave less frequently at secondary basic sites. Its most common secondary cleave site is between the Gly and Arg residues although the frequency of these events is protein specific.
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.
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