Plasmid Construction for Promoter, RBS, and Terminator Assay

All the plasmids used in this study are listed in Additional file 1: Table S1, which were constructed by the Gibson Assembly method [26 (link)], using linear fragments purified from PCR products. The promoter and terminator sequences from Additional file 1: Table S2 were amplified by PCR using Synechocystis 6803 genomic DNA as template. The RBS sequences were selected as 22-bp immediately preceding the translational start codon of each gene. The DNA fragments for construction of the plasmids pCA-UC118 and pCB-SC101 were amplified from Synechocystis 6803 genomic DNA, plasmid pUC118 [34 (link)], and plasmid pSC101 [35 (link)], respectively. All of the plasmids for assay of promoter, RBS, and terminator activities were ligated to the plasmid backbone pRSF1010, which is a derivative of the pPMQAK1 broad host range vector [6 (link)].
All PCR amplifications were performed using Phusion High-fidelity DNA polymerase (Thermo Scientific). Plasmids and PCR products were purified using the GeneJET (Thermo Scientific) plasmid miniprep kit and gel extraction kit, respectively. Oligonucleotides were designed using the SnapGene software (GSL Biotech LLC) and synthesized by IDT (Coralville, IA). All oligonucleotides used in this study are listed in Additional file 1: Table S3.