Figure 1

Brief outline of TREC and background formation. The gene to be inserted (your favorite gene, YFG), indicated by a purple arrow can be seamlessly inserted into a target site (orange arrow) via TREC. The knock-in sequence (purple arrow) is placed immediately downstream of the repeat sequence (RS) (dark blue box containing a black arrow) in the CORE cassette (gray boxes). After integration, induction of double strand break can promote homologous recombination between the two repeat sequences, leading to removal of the CORE cassette, as shown on the left. However, a fraction of transforming DNA may circularize itself through illegitimate recombination or non-homologous end joining, where broken DNA ends join. The resulting DNA may be maintained as a non-integrated plasmid if the knock-in sequence contains ARS activity, as shown on the right. UHR, indicates upstream homology region, and DHR, downstream homology region.