The advent of synthetic transcriptional regulators built mainly on human-derived proteins, namely synthetic Zinc Finger Transcription Regulators (synZiFTRs), has enabled fine-tuned control of therapeutically significant genes in primary T cells. However, their clinical relevance could be enhanced by amplifying synthetic gene circuit activation and expanding the synZiFTR toolkit with standardized compo-nents for the construction of more complex circuits. This study describes the de-velopment of the next iteration of synZiFTR, the synZiFTR2.0, incorporating the human-derived transcription elongation domain, IWS1. We present an engi-neered version 2.0 of GZV- and 4OHT/TMX-regulated gene switches, exhibiting a robust increase in transcriptional output upon drug induction. Furthermore, the synZiFTR toolkit was expanded and utilized to examine the feasibility of con-structing a two-input AND logic gate. Interestingly, the integration of IWS1 un-veiled a potential role of PP1-NUTS phosphatase in enhancing synthetic circuit output, though the precise mechanism warrants further investigation. The intro-duction of synZiFTR2.0 is projected to boost its clinical applicability, particularly in settings where circuit output strength is contingent on disease context that is often uncertain. / 2025-10-03T00:00:00Z