Despite recent advances in improving titers for therapeutic proteins such as antibodies to the 10 g/L scale, these high yields can only be achieved in select mammalian hosts. Regardless of the host or product, strong promoters are required to obtain high levels of transgene expression. However, the promoters employed to drive this expression are rather limited in variety and are usually either viral-derived or screened empirically during vector design. To begin to move away from viral parts, we employed a more systematic approach to identify and design new synthetic promoters using endogenous elements. To do so, we established a workflow to design these elements by (1) analyzing the transcriptomics profile of a specific cell line under a desired, representative cell culture condition, (2) identifying key genetic motifs using bioinformatics that can be used to rationally construct synthetic promoters, (3) building synthetic promoters using conventional DNA synthesis and molecular biology techniques, and (4) evaluating the performance of these synthetic promoters using model proteins. The resulting promoters perform comparably to the hCMV IE promoter variants tested, but with endogenous components. During this design-build-test cycle we also investigated the underlying design rules for transcription factor binding site arrangement in synthetic promoters. Overall, this approach of using an "omics-guided" workflow for designing synthetic promoters facilitates the construction of high expression vectors for immediate use in current production hosts.