YFLEX: Saccharomyces cerevisiae ORF Clone Resource

描述（由申请人提供）： 这项工作的总体目标是以重组克隆形式（在这种情况下，来自Invitrogen的Gateway克隆系统）创建酿酒酵母ORF的全面的、序列验证的克隆集合，称为YFLEX（酵母全长表达）ORF克隆文库。重组克隆方法允许将整个ORF Entry克隆文库移动到新的表达质粒中，从而促进该文库在许多不同载体和实验形式中的使用。正因为如此，我们期望YFLEX文库和由其创建的表达文库将成为酵母研究界的宝贵工具，并有可能成为更广泛的研究界。YFLEX克隆将通过蛋白质组学研究所（HIP）和Invitrogen提供。 该提案的具体目标是： 1.完成YFLEX ORF Entry克隆文库。 在YFLEX项目的这一阶段，需要克隆大约2000个ORF，以提供S.酿酒酵母基因组这将包括最近重新定义的518个ORF。我们还将用无内含子克隆替换225个含有内含子的YFLEX克隆。 2. YFLEX ORF Entry克隆的序列验证。 将对YFLEX克隆文库中的所有ORF克隆进行全长序列验证，以确保每1000个碱基最多有一个错误。在完全测序后，当不存在导致保守氨基酸取代的超过一个序列差异，并且不存在导致非保守取代或导致过早ORF截短的差异时，克隆将被视为完成。

DESCRIPTION (provided by applicant): 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.