Schematic representation of TREC-IN. (A) Outline of the TREC-IN method. Step 1: The target region (orange arrow) is replaced with a CORE6 cassette by homologous recombination via two 50-bp homology sequences [upstream homology region (UHR) or repeat sequence (RS), indicated by dark-blue box containing a black arrow, and downstream homology region (DHR), indicated by light-blue box]. Transformed cells were selected for Uracil prototrophy. The cassette includes the reconstituted I-SceI binding site (black bar), and the I-Sce1 gene under the control of the Gal1 promoter, a Uracil marker, and 5’ kanMX gene componenet. Step 2: The knock-in module [containing your favorite gene/ sequence (YFG/S, purple arrow) and the 3’ region of the kanMX gene component, interspersed by the 50 bp RS, and flanked by 50 bp homology region to the 5’ kanMX gene component in the cassette and DHR to the target site] is transformed and selected on geneticin resistance for full complementation of the kanMX gene component. Step 3: Yeast colonies are grown in presence of galactose to express I-SceI, which produces a double-strand break at the I-SceI site, and enhances homologous recombination (dashed double-headed arrow) between the two RS, resulting in excision of the CORE6 and knock-in modules. Colonies are grown on 5-FOA for Uracil counter selection. Primers to confirm correct insertions are shown by dashed arrows, and synthetic primers are represented by kinked arrows. (B) CORE6 cassette construction. The CORE6 cassette is amplified from pCORE6 to add four nucleotides (tagg) for full reconstitution of the I-SceI binding site (black bar). 50 bp UHR and 50 bp DHR, specific to the target site, are also included in the construction. (C) Knock-in module construction. The knock-in module is constructed by a PCR-based fusion method. The two amplicons are the 3’ kanMX gene component carrying the 3’ region of kanamycin gene (gray striped box), the RS, and a homology region to the 5’ region of the kanamycin gene in CORE6 (gray box), and the replacement gene (YFG/S) flanked by the RS and DHR.