Abstract
The capacity of Saccharomyces cerevisiae to repair exposed DNA ends by homologous recombination has long been used by experimentalists to assemble plasmids from DNA fragments in vivo. While this approach works well for engineering extrachromosomal vectors, it is not well suited to the generation, recovery and reuse of integrative vectors. Here, we describe the creation of a series of conditional centromeric shuttle vectors, termed pXR vectors, that can be used for both plasmid assembly in vivo and targeted genomic integration. The defining feature of pXR vectors is that the DNA segment bearing the centromere and origin of replication, termed CEN/ARS, is flanked by a pair of loxP sites. Passaging the vectors through bacteria that express Cre recombinase reduces the loxP-CEN/ARS-loxP module to a single loxP site, thereby eliminating the ability to replicate autonomously in yeast. Each vector also contains a selectable marker gene, as well as a fragment of the HO locus, which permits targeted integration at a neutral genomic site. The pXR vectors provide a convenient and robust method to assemble DNAs for targeted genomic modifications.