Patent ID: 6207442
Filing Date: 2001-03-27
Classification: C07K,C12N

Abstract:
A method for preparing a double stranded, circular DNA molecule comprising the steps of:(a) providing a double-stranded donor DNA fragment encoding a polypeptide of interest;(b) providing a double-stranded, linear acceptor plasmid having blunt first and second ends and comprising a selectable marker and replication sequence that are functional in Saccharamyces cerevisiae, wherein the acceptor plasmid is essentially free of DNA encoding the polypeptide of interest;(c) providing a first double-stranded DNA linker comprising a first segment identical in sequence to a first region of the acceptor plasmid and a second segment identical in sequence to a first region of the donor DNA fragment, wherein each of the first and second segments of the first linker is at least 10 bp in length;(d) providing a second double-stranded DNA linker comprising a first segment identical in sequence to a second region of the acceptor plasmid and a second segment identical in sequence to a second region of the donor DNA fragment, wherein each of the first and second segments of the second linker is at least 10 bp in length; and(e) combining the donor DNA fragment, acceptor plasmid, first DNA linker, and second DNA linker in a Saccharomnyces cerevisiae host cell whereby the donor DNA fragment is joined to the acceptor plasmid by homologous recombination of the donor DNA, acceptor plasmid, and linkers to form a closed, circular plasmid comprising a region encoding the polypeptide of interest.