A three-step denaturation, annealing, and extension protocol was used for both mutants with an annealing temperature of 67.6°C for F104 and 72.0°C for F121Y. All PCR experiments were performed using a Axygen Maxygene II instrument. All PCR products were transformed into DH5α cells (Thermo Scientific) for plasmid expression. Single mutant DNA sequences were confirmed using Sanger Sequencing at the UMN Genomics Center.