The overall goal of this effort is to create a comprehensive, sequence-validated clone collection of Saccharomyces cerevisiae ORFs in a recombinational cloning format (in this case, the Gateway cloning system from Invitrogen), termed the YFLEX (Yeast Full Length EXpression) ORF clone library. A recombinational cloning approach allows an entire ORF Entry clone library to be moved to a new expression plasmid and thus facilitates the use of that library in many different vectors and experimental formats. Because of this, we expect that the YFLEX library and the expression libraries created from it will become invaluable tools for the yeast research community, and, potentially, the wider research community. The YFLEX clones will be made available through the Institute of Proteomics (HIP) and Invitrogen.
The Specific aims of this proposal are: 1. Completion of the YFLEX ORF Entry clone library. In this phase of the YFLEX Project, approximately 2000 ORFs will need to be cloned to provide complete representation of the 5600 ORFs encoded in the S. cerevisiae genome. This will include 518 ORFs that have been recently redefined. We will also replace 225 YFLEX clones containing introns with intron-less clones. 2. Sequence validation of the YFLEX ORF Entry clones. All ORF clones in the YFLEX clone library will be full-length sequence validated to at most one error per 1000 bases. Clones will be accepted as finished when, after full sequencing, there is no more than one sequence discrepancy that results in a conservative amino acid substitution, and there are no discrepancies resulting in non-conservative substitutions or which lead to premature ORF truncation.
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