2.2. RNAi Expression Vector Construction

The pSCTGA vector, containing a Km resistance cassette with mobilization (Mob) genes, was constructed based on the plasmid RSF1010 [24], and can successfully replicate in Synechocystis 6803. The pSCTGA vector was modified for use as an RNAi expression vector in Synechocystis 6803. Firstly, the pSCTGA vector backbone (pSCTGA-backbone) containing the replication elements, Mob gene, and Km resistance gene was cloning using the primer pair, pSCTGA-backbone-F and pSCTGA-backbone-R (Table S1). The forward and reverse sequences of P_cpcB (FP_cpcB and RP_cpcB) were PCR amplified from the WT Synechocystis 6803 genome using the primer pairs, FP_cpcB-F + FP_cpcB-R and RP_cpcB-F + RP_cpcB-R, respectively (Table S1). The multiple cloning site (MCS) was PCR amplified from L4440 vector [10] using the primer pair, MCS-F and MCS-R (Table S1). Next, the FP_cpcB, MCS and RP_cpcB DNA fragments were jointed together to form a recombinant DNA fragment (FP_cpcB-MCS-RP_cpcB) using fusion PCR with the primer pairs, FP_cpcB-F + MCS-R and FP_cpcB-F + RP_cpcB-R (Table S1). Finally, the FP_cpcB-MCS-RP_cpcB DNA fragment was joined to the pSCTGA-backbone in a correct orientation via the one-step cloning method to form the pSCT3C RNAi expression vector.