Significance A central question in biology is how transcription factors (TFs) recognize specific binding sites in enhancers and regulate gene expression. In general, only a fraction of potential binding sites for TFs are occupied in a particular cell type. TF affinity for a motif site, local interactions among TFs, and larger-scale chromatin accessibility can influence binding, although the relative contributions of these factors is unclear. Moreover, little is known about how specific combinations of TFs control quantitative gene expression once bound. Here, we use large-scale synthetic biology approaches to explore the features that govern TF binding vs. enhancer activity. This approach provides a paradigm for systematic study of key regulatory sequences within enhancers and how they interact to influence gene expression.