Evidence item

Here, we develop a library of synthetic promoters (SPs) responsive to seven TFs identified through transcriptomic analysis of T-cell activation programs. These promoters couple endogenous TF activity to transcriptional output across a broad dynamic range of inducibility and basal expression. We quantitatively profile promoter performance in HEK293 and Jurkat cells, evaluate them for orthogonality, and integrate them into Boolean logic gate architectures and network topologies to minimize basal transcriptional leakiness and improve ON/OFF ratios. We test selected synthetic promoters in primary human CD8⁺ T cells, under endogenous TF regulation, and we further characterize the NR4A2-responsive promoter subset in vitro and in tumor-infiltrating engineered T cells in a melanoma xenograft model, demonstrating dynamic and reversible activation.