The FD-CF synthesized fluorescent proteins were expressed at 30°C (for the constitutively expressed outputs) or 37°C (for the toehold-encoded designs) overnight and visualized using a Safe Imager 2.0 Blue-Light Transilluminator (Thermo Fisher Scientific), white light, or the inexpensive imager developed as part of the BioBits™ kit. Images were taken with a DSLR camera and adjusted and cropped in Adobe Photoshop. For quantitative analysis, cell-free reactions were transferred to a 384-well clear-bottom, black-walled plate, and relative fluorescent units were read on a SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices).

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