The 5′-terminal end of the forward primer DNATFBS-F0 was labeled by biotin (table S1). The biotin-labeled DNA sequences containing intact TFBS of HosA (DNAHosA-0) were generated by annealed primers (DNAHosA-F0 and DNAHosA-R0). The biotin-labeled DNA sequences containing nicked TFBS of HosA (DNAHosA-N, where N indicates the nick position) were obtained by annealed primers (DNAHosA-F0, DNAHosA-RN-1, and DNAHosA-RN-2; table S1). Other biotin-labeled DNA sequences containing intact or nicked TFBS of TetR and AvaR1 were obtained in the same manner as for that of HosA. The primer annealing was performed by heating at 95°C for 5 min, followed by slowly cooling to room temperature. The generated bio-DNATFBS were verified by agarose gel electrophoresis and were quantified using a NanoVue plus Spectrophotometer (GE Healthcare). Kinetics between aTFs (HosA, TetR, and AvaR1) and their nicked TFBSs were determined by BLI assay using an Octet RED96 system (FortéBio). Briefly, the process consisted of five steps: balance, DNA loading, rebalance, association, and dissociation. The blank tests were carried out by using HBS-EP buffer instead of aTFs in the association step and used for baseline correction. kon, koff, and KD (KD = koff/kon) values were calculated by fitting the processed data (baseline correction and normalization) with a 1:1 model using the Octet Analysis System 21 CFR Part 11 (version 9.0). The regression coefficients (R2 value) and error values were used to assess the quality of the fits to the data.

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