Cell engineering presents great potential for next-generation biotechnological and biomedical applications that employ engineered mammalian cells to sense intracellular or extracellular signals and respond in a user-defined manner. Programming mammalian cells requires tools that operate orthogonally to the cellular machinery for sensing and controlling the levels, localization, and function of endogenous proteins. This work describes my efforts to expand the mammalian synthetic biology toolbox and develop orthogonal tools and gene circuits to reprogram cells for a diverse range of applications. I first illustrate the development of a gene signal amplifier platform to monitor chromosomal gene expression that recapitulates gene expression dynamics from the native chromosomal context and generates a readily detectable signal output. The gene signal amplifier links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal target gene. By recapitulating the transcriptional and translational control mechanisms underlying the expression of a target gene with high sensitivity, this platform provides a novel technology for monitoring target gene expression with superior sensitivity and dynamic resolution. To illustrate the utility of such a reporter system, I used the gene signal amplifier platform to monitor the activation and dynamics of the unfolded protein response (UPR), a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). The UPR manifests as a series of signaling cascades aimed at relieving proteotoxic stress and restoring homeostasis or executing apoptosis to eliminate irremediably damaged cells. To control cellular fate upon UPR induction, typically caused by overexpression of recombinant proteins, I designed a series of orthogonal genetic circuits that interface with the UPR, sense and integrate signals associated with activation of the UPR and, in response remodel the stress response pathway to enhance stress attenuation and delay apoptosis. Finally, I developed a nanobody-based platform for controlling the selective localization of a target protein to multiple cellular compartments and investigated different circuit architectures to obtain dynamic control over the localized target protein. Combining nanobody-mediated localization and degradation with orthogonal regulation, I generated a nanobody-based platform to control the relative concentration and localization of a target protein in cells. The tools described in this work are expected to have a significant impact on the design of strategies to reprogram mammalian cells for a diverse range of applications including the development of cell-based therapies and the production of biologics.