FLAG tag module for PCR based gene targeting

Abstract

Epitope tagging of yeast proteins has become an efficient tool for biochemical analysis of protein of interest. The epitope-tagged proteins can be used for western blotting, immunoprecipitation and immunofluorescence experiments without the need to raise specific antibodies, thus saving considerable time and expense. We have constructed plasmid containing FLAG tag with kanMX6 module, which allows selection of G418-resistant cells in yeast. The same set of primers that amplify module constructed by Bahler et al. can be used to amplify the FLAG tag module constructed in this study. The linear DNA fragment containing FLAG tag module with flanking homology region of gene of interest can be efficiently integrated on the yeast genome using homologous recombination. We have successfully FLAG tag wat1/pop3 gene at its chromosomal locus and confirmed by western blot analysis. This construct can be of very useful for generating C terminal tagging of desired genes at its normal chromosomal locus without interfering their function.