Construction of GFP-reporter plasmids with 5′UTRs

The tetracycline-inducible promoter with GFP construct (pEZ-tet-GFP) was derived from the minimal shuttle vector pEZ15Asp of Z. mobilis and contains the replication origin of E. coli and Z. mobilis (Yang et al., 2016b). A parental plasmid containing pheS counter-selection marker (Kast, 1994; Miyazaki, 2015) was constructed with pheS counter-selection marker incorporated in front of the gfp gene flanked by BsmBI sites (Type IIS enzyme), which is one of the Type IIS restriction endonuclease to enable Golden Gate cloning for efficient cloning of each 5′UTR-containing GFP (Engler et al., 2008; Figure ​Figure3E).3E). With this design, 5′UTRs along with the first 90-bps of the downstream mRNA were cloned for each candidate right in front of the gfp gene in frame. This design was implemented to preserve the native structure of the UTR by better mimicking its native context while still allowing GFP expression. Primers used for the amplification of UTR+90-bps are listed in Table S3. Each primer contains a BsmBI enzyme site on the 5′ end.