###### Abstract
Temperature-dependent melting of hairpin secondary structure exposes the ribosome binding site (RBS), and protein translation can occur. RNA itself can serve as an input to control RNA devices via Watson-Crick base-pairing rules. For instance, cis-repressor forms base pairing with RBS to occlude translation initiation. The interaction of cis-repressor and trans-acting RNA induces a conformational change of cis-repressor such that the RBS is accessible and protein translation can be initiated (Figure 1b) [16]. Small molecule and protein-responsive RNA control devices have been engineered by coupling aptamers to gene regulatory components due to their high affinity and specificity to target molecules [27; 28]. For example, an aptazyme, combining a theophylline RNA aptamer to a hammerhead ribozyme, represses downstream gene expression by sequestering RBS within the ribozyme stem (Figure 1c) [29]. When theophylline is available, the ligand-aptamer interaction induces a conformational change of ribozyme to induce cleavage of its stem, thereby allowing translation of the downstream gene. De novo-designed synthetic RNA regulators, small-transcriptional activating RNA (STAR) and toehold switch (THS), which each operate at transcription and translation steps, offer promising toolbox with large library size and high dynamic ranges rivalling those of well-known protein regulators [30; 31]. For example, a library of 73 TetR-family repressors showed 5- to 207-fold change with up to 16 orthogonal repressor sets [32]. De novo-designed RNA regulators, STAR and THS, demonstrated libraries with comparable dynamic range and orthogonality. STAR system is composed of a target RNA placed upstream of a gene that, once transcribed, folds into a rho-independent transcription terminator, preventing transcription of the downstream gene (Figure S1a) [30]. Upon interaction between target RNA and cognate antisense RNA, the terminator formation is prevented, allowing transcription of the downstream gene. The STAR library was further expanded through computational design [33], applied to plant viral RNA detection [34], and switchable feedback regulators [35]. However, the STAR library was created by randomizing only the upstream linear toehold sequences rather than the stem-loop sequence of terminators; in that sense, the design flexibility of the STAR system remains underexplored. On the other hand, THS is a riboregulator that regulates gene expression at the translation stage (Figure S1b). THS demonstrated high ON/OFF ratios and straightforward concatenation and expansion towards complex logic evaluation; however, some leaky expressions were observed even in the absence of trigger RNA, possibly due to the presence of complete RBS within THS RNA [31; 36]. The presence of leaky expression may require further containment in order to construct complex multilayered circuit architecture [37], and consequently, recent studies aimed to demonstrate tight leakage control by simultaneous regulation of transcription and translation steps [38; 39; 40; 41; 42]. Here, inspired by STAR library construction, we devise novel switchable transcription terminators (SWT) by modifying natural and synthetic terminators with additional engineered toehold sequences. While the STAR library focused on randomization of the linear toehold region upstream of the fixed terminator sequence, SWT was designed to explore a different set of terminator sequences and also the sequence variants of a given terminator to further expand the sequence space of RNA transcription regulators. Further, by combining the new SWT with well-characterized THS, we demonstrate a multilevel regulatory system at both transcription and translation steps. This multilevel regulator showed tight leakage control in _E. coli_ and could function as 2-input AND logic gates. In addition, SWT and THS can be flexibly combined for controlling multiple fluorescent proteins and could prove useful in multi-layer circuits in vivo.