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Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, a