Genomic Promoter Shuffling by Using Recyclable Cassettes

Genetic elements of interest can be introduced into the Saccharomyces cerevisiae genome via homologous recombination. A common method is to link such an element to a selectable marker gene to be integrated into the target locus. However, the marker gene in this method cannot be reused, which limits repeated manipulation of the yeast genome. More importantly, it cannot be conveniently used to integrate a promoter element. An alternative method is to utilize a counterselectable gene, such as URA3, with flanking tandem repeats. After integration, URA3 along with one copy of the repeat can be popped out via internal recombination, leaving behind one copy of the unwanted repeat. Here we describe a method of genetic element shuffling in which the tandem repeats are made of a set of promoters, so that after integration and popping out, only one copy of the promoter remains at the desired locus to function.