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Figure S1. The numerical summary of nucleotides at 80 randomized positions. Nucleotide frequency of (a) 40 random clones in original promoter library and (b) 20 clones isolated by FACS screening. Boxed sequences represent two invariable site—RBS “AGGA” and BamHI site (GGATCC). In (b), the conserved sequence (GTAnnaTnG) was shown in red-color letters and underlined.

Figure S2. Mean fluorescence intensities of all 80 isolated clones.

Figure S3. Correlation between GFP expression level (x-axis), mRNA transcript level (y-axis) and mean fluorescence intensity (z-axis). The measured data were shown in Table S3.

Figure S4. Activity of 20 synthetic promoters in E. coli. a: GFP fluorescence intensities of the synthetic promoters in pCES208 derivatives with GFP in E. coli were analyzed by flow cytometry. E. coli (pCES208) was used as negative control. b: The level of GFP expression in each E. coli clone was analyzed by Western blotting. Arrow indicates GFP (∼30 kDa).

Figure S5. Activity of two synthetic promoters (PH30 and PH36) in E. coli under IPTG induction (left) and no induction (right). The fluorescent intensities of the E. coli harboring pET22b (red), pET22b-T7-GFP (cyan), pET22b-H30-GFP (yellow), and pET22b-H36-GFP (green) were analyzed by flow cytometry.

Table SI. List of PCR primers used in this study.

Table S2. Summary of FACS screening of synthetic promoter library.

Table S3. The three different measures (mean fluorescence value by FACS, quantification cycle by qRT-PCR, GFP expression level by Western blotting) from the 20 synthetic promoters and trc promoter.

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