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None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. is the so-called 50:50 method (Horecka and Davis, 2014), which allows for gene deletion without a heterologous sequence via a single PCR step, followed by transformation and subsequent counter-selection. The outstanding feature of this method is the use of 50:50 primers that are designed to direct a pop-out recombination event just downstream of the upstream pop-in site. The heterologous sequences introduced by pop-in recombination are then excised by the pop-out recombination, leaving no traces. This method is also applicable for the insertion of a codon, site-directed mutation, as well as deletion of two nucleotides for introducing a frameshift mutation (Horecka and Davis, 2014). However, there is currently no method for epitope tagging based on the 50:50 strategy. Here, we have constructed a new set of plasmids for the C-terminal tagging of epitopes with no extraneous sequences other than the epitopes on the basis of the 50:50 method. The modules used for PCR-mediated epitope tagging are shown in Fig. 1A. Starting from the plasmid pF6a3xHA-His3MX6 (Longtine et al., 1998), which is widely used for the insertion of the 3xHA epitope, two modifications were introduced. First, we replaced the His3MX6 marker with the GAL10p-GIN11M86-LEU2 cassette (Akada et al., 2002) to introduce a counter-selection marker, resulting in the plasmid pMN-12. The GAL10pGIN11M86 DNA sequence inhibits cell growth when overexpressed from the GAL10 promoter (Akada et al., 2002). Therefore, pop-out recombinants can be selected in media that contain galactose as a carbon source. LEU2 is used as a transformation marker. Second, to expand the utility of the plasmid, we replaced the DNA sequence encoding the 3xHA epitope in pMN-12 with those for dual tags in which a single HA epitope was tagged with each of 13xMyc and 3xFLAG, resulting in the plasmids pMN13 and pMN-15, respectively (Fig. 1A, and see below). Marker-free insertion of a series of C-terminal epitopes based on the 50:50 method in Saccharomyces cerevisiae

Marker-free insertion of a series of C-terminal epitopes based on the 50:50 method in <i>Saccharomyces cerevisiae</i>