JCVI-syn3B FtsZ:mCherry plasmid

This plasmid was used for genetic introduction of a second copy of the FtsZ gene fused with mCherry to JCBI-syn3B for fluorescent imaging of cell division. Gibson assembly was used to construct the plasmid. Syn1.0 (GeneBank accession number CP002027) served as the template to amplify fragment 1 using primers 5’-GAATTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCTTTTAAAAATGTCGGAAAGTCATC-3’ and 5’-AGCAAAGTGGGTGATAAATAAATGACAAACGAATTTAAACAAATAGC-3’, and fragment 2 using primers 5’-CGTTTGTCATTTATTTATCACCCACTTTG-3’ and 5’-ATTTGAACGTTGCGAAGCAACAGAAGCATAATAACAATTATTAAT-3’. The vector backbone was amplified using primers 5’-AAGGATCCGCTGGCTCCGCTGCTGGTTCTGGCGAATTCATGGTATCAAAAGGAGAAGAAGATAATATG-3’ and 5’-TAATTGTTATTATGCTTCTGTTGCTTCGCAACGTTCAAATC-3’, with the ptxB-FLAG plasmid (not published) as the template. PrimeSTAR Max DNA polymerase (Takara, #R045A) was used to amplify the fragments. PCR was performed with an initial denaturation at 98 °C for 3 minutes, followed by 30 cycles of 98 °C for 10 seconds, 55 °C for 10 seconds, and 72 °C for 1 minute, with a final extension at 72 °C for 5 minutes. Primers 5’-TCCTCCAGCTCCTAATCCTT-3’ and 5’-TGTTTGTCGGTGAACGCTCT-3’ were used for colony PCR to identify positive clones. The final plasmid was confirmed by sequencing.