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An Epitope Tagging Vector for Gene Expression in Mammalian Cells

of the target protein, and highly immunoreactive. The FLAG epitope is a synthetic epitope that consists of eight amino acid residues (DYKDDDDK).5 The c-myc epitope is derived from the human c-myc gene and contains 10 amino acid residues (EQKLISEEDL).6 In addition to the epitope tag sequences, the pCMV-Tag1 vector contains features for expression of fusion proteins in eukaryotic cells. The cytomegalovirus (CMV) promoter allows constitutive expression of the cloned DNA in a wide variety of mammalian cell lines. The neomycin-resistance gene is under control of both the prokaryotic b-lactamase promoter to provide kanamycin resistance in bacteria and the SV40 early promoter to provide G418 resistance in mammalian cells. The multiple cloning site (MCS) of the pCMV-Tag1 vector is arranged to allow a variety of cloning strategies to be used, resulting in C-terminal, N-terminal or internal tagging of the protein of interest. A Kozak consensus sequence of GCC(A or G)CCATGG7 provides optimal expression of the fusion protein when the N-terminal FLAG epitope tag is used. Other cloning options, which require fusion proteins to include their own translational start sequence, are also possible.

Cloning Strategies for Epitope Tagging

Epitope Tag

Tag Location

Cloning Site

Kozak Sequence
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