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Background  Recombinatorial cloning using the Gateway TM  technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in Gateway TM  compatible vectors. The MultiSite Gateway TM  system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast  Saccharomyces cerevisiae , one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.    Results  Here, we present a set of three-fragment MultiSite Gateway TM  destination vectors that have been developed for gene expression in  S. cerevisiae  and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins.    Conclusion  Our vectors make MultiSite Gateway TM  cloning accessible in  S. cerevisiae  and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research.

A MultiSite GatewayTM vector set for the functional analysis of genes in the model Saccharomyces cerevisiae