Rational design of intrinsic transcription terminators with programmable strength

Intrinsic terminators are widely used in synthetic biology and metabolic engineering, yet their strength must be selected empirically from characterized libraries. We therefore developed a framework for designing intrinsic terminators with programmable strength. TK, designed to maximize termination strength, exceeded both B0010, a widely used standard, and the strongest terminator in a 582-member reference library when assayed in parallel. To establish that these gains reflect intrinsic termination rather than cellular context, we developed a cloning-free in vitro transcription assay using fluorescent light-up aptamers; TK again ranked highest. The framework improved the canonical T7 terminator and recovered strength rankings across three other bacterial species. Designs were generated using an interpretable sequence-to-function model trained from primary-sequence descriptors alone. The framework is available as open web tools, TerSP for strength prediction and TerFac for target-driven design. Model interpretation revealed previously unreported determinants, including a preference for compositionally diverse hairpin stems.