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In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2‐μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir0 strain as a host for constructing an entire 2‐μm plasmid with an expression construct using the three‐fragment gap‐repair method without Escherichia coli...