Gibson assembly 2 master mix

All plasmids were constructed by Gibson Assembly 2× Master Mix (NEB); all PCR products were generated using Q5 Hot Start 2× Master Mix (NEB). E. coli strain S1030^{30 (link)} were transformed by electroporation with three plasmids: (i) a complementary plasmid (CP) that constitutively expresses a PA-RNAP with one of the three protease cut sites (Supplementary Fig. 2), (ii) an accessory plasmid (AP, Supplementary Fig. 4) that encodes gIII-luciferase (translationally coupled) under control of the T7 promoter, and (iii) an arabinose-inducible expression plasmid for one of the three proteases (EP, Supplementary Fig. 3). The HRV protease gene was purchased as IDT gblocks and cloned into the expression vector. The MBP-TEV fusion protein was amplified by PCR from pRK793^{41 (link)}. The MBP fusion was necessary for expression and solubility. We deployed a constitutively active HCV protease construct that includes the NS4a cofactor peptide^{42 (link)}. Cells were grown in 2xYT media to saturation in the presence of antibiotics and 1 mM glucose, then inoculated into 1 mL fresh media containing 1 mM glucose and antibiotics in a 96 well culture plate. After 4.5 h, 150 µL of the cultures were transferred to a black-wall clear-bottom assay plate and luciferase and OD₆₀₀ measurements were taken using a Tecan Infinite Pro plate. The luminescence data was normalized to cell density by dividing by OD₆₀₀.