Cloning and Mutant Generation of Plant Genes

Cloning of CrPAS (MH213134), CrDPAS (KU865331), CS (MF770512) and TS (MF770513) was reported in Caputi et al.² . Cloning of DPAS and CorS from Tabernanthe iboga was reported in Farrow et al.⁶ . CS, TS and CorS mutants were generated by overlap extension PCR. The codon(s) to be mutated was selected and two primers, one reverse and one forward (Supplementary Table 3), were designed to overlap and introduce the mutation. PCR products were gel purified, ligated into pOPINF expression vector^{19 (link)} using the In-Fusion cloning kit (Clontech Takara) and transformed into competent E. coli Stellar cells (Clontech Takara) according to the manufacturer’s instructions. Mutant constructs were sequenced to verify the mutant gene sequence and correct insertion.