The complete sequencing of the Saccharomyces cerevisiae genome provides a powerful tool for studying and elucidating essential cellular processes. To aid in the application of this resource to investigations into the molecular mechanisms of endoplasmic reticulum-associated protein degradation, a simple procedure was designed to generate a unique 2-microm LEU2-selectable yeast expression vector. Putative genes easily inserted into this vector are under control of the ADH1 promoter and transcription terminator sequences. Furthermore, a LEU2 selection in both yeast and Escherichia coli was used to allow the isolation of underrepresented plasmid from a pool of multiple vectors. Together, these advances in technology will be useful in the systematic analysis of novel yeast gene function.