in vitro synthesis of α-factor, native CALB and codon-optimized CALB genes.

A single-step strategy (A-PCR) was conducted to synthesize the codon-optimized α-factor (A and B), and a two-step strategy combining A-PCR and OE-PCR (C) was conducted to synthesize the native CALB (D) and codon-optimized CALB (E) genes. In order to synthesize the native CALB, the oligonucleotides were firstly assembled into F1 (541 bp) and F2 (510 bp), and then they were assembled into the genes with native signal peptide (CalBSP), native pre-sequence (CalBP) and mature CALB (CalB) with different primer pairs at OE-PCR step (D). In order to synthesize the codon-optimized CALB, the oligonucleotides were firstly assembled into F1M (510 bp) and F2M (553 bp), and then they were assembled into genes with signal peptide (CalBSPM), pre-sequence (CalBPM) and mature CALB (CalBM) with different primer pairs at OE-PCR step (E).