Single cell organisms are masters at adapting to a broad spectrum of environmental stimuli. Following an initial signal, the first proactive response of the cell is often the transcription of genes. In the model eukaryote, yeast, transcription is regulated by more than 200 factors that can bind in any combination to the regulatory sequences of genes. It is speculated that this complexity allows the organism fine control and feedback over the activation or repression of transcription. Computational analysis, alone, has resulted in a number of regulatory structure models. However no single model has proven predictive. We will describe experimental efforts to combine computational predictions, classical genetics, and functional genomics with new technologies in synthetic biology to query regulatory architecture.