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From 2010.igem.org

Yeast: A Simpler Transformation

Yeast Vectors

Two new plasmid backbones are to be created and submitted to the registry. Similar in structure the two vectors will be derived from biobrick [http://partsregistry.org/wiki/index.php?title=Part:pSB1AT3 pSB1AT3] which contains a high copy E. coli replication origin and both ampicillin and tetracycline resistance. PCR will be use to modify the site upstream of the prefix and downstream of the suffix in vector. The Plasmid will take the following design:

This allows the insertion of yeast biobrick parts (including positive selection markers) into the vector using E. coli. Once the construct is fully assembled the vector is linearized with BbsI and transformed into yeast. BbsI was chosen since it cuts downstream of its recognition site, and will create non-overlapping ends preventing circularization of the linear products. The two new vectors will contain sequences homologous to the Ura3 and His3 loci in the yeast genome. When employed either the Ura3 or His3 loci will be replaced with the construct, creating an autotrophic strain of yeast. When coupled with a positive selection marker this will allow for both positive and negative selection of transfomants.