Targeted Integration of Complex Genetic Elements at Multi-Copy Loci by Golden Gate Assembly

Budding yeast Saccharomyces cerevisiae has become a model eukaryotic microorganism for targeted genomic manipulation due to its efficient homologous recombination. A few genomic loci, including rDNA, Delta, and Ty1, can be utilized to introduce variable copies of genetic elements into the yeast genome. Here we describe a method that combines in vitro Golden Gate Assembly to assemble one or a complex genetic element in an orderly manner and then integrate it into predetermined multi-copy loci through homologous recombination. Different transformants may contain different copy numbers, which allows the selection of desired levels of target gene expression.