A versatile toolbox for PCR‐based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes

TLDR

PCR‑based chromosomal integration of amplified cassettes is a fast, widely used method for tagging proteins in *Saccharomyces cerevisiae*, allowing precise insertion of any sequence at a chosen locus via homologous primers. The authors constructed 76 novel cassettes that combine diverse C‑terminal epitope tags, nine promoter substitutions, and N‑terminal tags to enhance the versatility of yeast gene tagging. These cassettes, which include the brightest yeast‑optimized red fluorescent protein RedStar2, additional fluorescent proteins, purification tags, new selection markers, and can be used for N‑ or C‑terminal tagging or gene deletion with only four primers, provide a cost‑effective and reproducible toolbox. The toolbox is expected to accelerate single‑gene and systematic studies of gene function in yeast. © 2004 John Wiley & Sons, Ltd.

Abstract

Abstract Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae . This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR‐primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C‐terminal tagged genes or for the exchange of the promoter and N‐terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C‐terminal epitope tags as well as nine different promoter substitutions in combination with N‐terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast‐optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N‐ and C‐terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost‐effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches. Copyright © 2004 John Wiley & Sons, Ltd.