Modular Scalable Synthetic Gene Circuits for Complex Functions Within ‎Minimal Computational Layers in Human Cells

Engineering mammalian cells to execute complex genetic programs remains a significant challenge in ‎synthetic biology. Synthetic gene circuits typically implement sophisticated programs through cascaded ‎computational layers, but these architectures require numerous orthogonal parts, increase genetic ‎payload, and deplete cellular resources, which limits functionality and scalability.‎‏ ‏We developed a modular design ‎framework to engineer scalable gene circuits that execute complex functions within fewer ‎computational layers. The platform integrates a toolkit to generate orthogonal trans-splicing-based ‎AND gates, native-synthetic hybrid promoters for tunable regulation, and synthetic microRNAs that ‎implement inhibitory logic. Using this approach, we engineered a three-input combinatorial logic gate, ‎a half adder, a full adder, and a dynamic 3-to-1 multiplexer with a dedicated Selector Overload Status ‎output, activated only when both selector inputs are activated. By minimizing computational layers while maintaining function, this strategy addresses scalability barriers in gene circuit engineering and expands opportunities for clinically relevant programmable therapies, biotechnology, and fundamental biology.‎