( of the target protein and h...)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