2.5.1. Overlapping DNA amplicons

The Kan resistance cassette was amplified by overlap PCR from the template plasmid pKD4 and hybrid primers. Primers were synthesized (Thermo Fisher Scientific) with 20 nucleotides of pKD4 priming sites and with 50 nucleotides from each side of the target genes (Supplementary Table S1). Extension of overlapping DNA amplicons was performed in a CFX96 PCR system (BioRad, Marnes-la-Coquette, France) with the following program: an initial denaturation of 30 s at 98°C; then 35 cycles of 10 s at 98°C, 30 s at 56°C, and 120 s at 72°C. The PCR products were purified with the Wizard SV Gel and PCR Clean-Up System (Promega), digested with DpnI (Thermo Fisher Scientific), re-purified, and finally suspended in sterile DNAse-free water.