SOE by PCR
SOE by PCR was carried out using a T100 Thermal Cycler (Bio-Rad,
Hercules, CA, USA) for 20 cycles, each of 1 min at 98°C, 1 min at 55°C,
and 2 min at 70°C. The reaction was carried out in a 50 µL volume
containing 2.5 U Phusion High-Fidelity DNA polymerase (Pfu; Thermo
Fisher Scientific), 200 µM dNTPs, 1 µL of primer mix (1.5 pmol per
primer), and 5 µL of 10× Pfu buffer.