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Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as transcriptional valves which can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly we construct 1183 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to simultaneously characterize the entire pool at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate their function using nearby sequence context. This work provides new avenues for controlling transcription and demonstrates the value of long-read sequencing for exploring complex sequence-function landscapes.
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