Extended Data Fig. 1: Characterizing, analyzing, and optimizing SENTR performance.

a, In vivo fluorescence of 1369 combinations of 5′ RNA-3′ RNA pairs. All 1296 combinations of 5′ RNAs with 36 5′ EGSs and 3′ RNAs with 36 3′ EGSs were validated, and another 73 groups of the 5′ RNA without 5′ EGS and 3′ RNAs without 3′ EGS were also characterized as control groups. b, SENTR performance with different lengths of EGSs. Different lengths of EGSs were designed by NUPACK with three trials, and the best-performing one of each length was shown. c, SENTR performance with EGS length gradient. The 50 nt EGS was shortened by every 10 nt (upper panel) and then every 5 nt (lower panel). d, ON state and OFF state fluorescence (upper panel) and ON/OFF ratios (lower panel) of 30 nt EGS library. e, The effect of 5′ spacer and 3′ spacer on SENTR performance. The 3′ spacer was designed to form P10 or not with the 3′ exon. Different lengths of 5′ spacers and 3′ spacers were tested. Error bars, mean values ± SD (n = 3). RPU, relative promoter units. Data in (a-e) were collected by flow cytometry. For (d), ON-state fluorescence was measured from cells expressing 5′ RNA and 3′ RNA, and OFF state fluorescence was determined from cells expressing only 3′ RNA. For (b-d), bars show mean values and error bars represent s.d. of n = 3 biological replicates. For (a, e), the heat maps show mean values of n = 3 biological replicates. Bacteria transformed with empty vectors and J23101-sfGFP were used as negative and positive controls for calculating fluorescence values in relative promoter units (RPU).