Abstract: Complex behavior within eukaryotic cells manifest from layered regulatory networks changing the transcription of many genes. To systematically study these pathways by modulating individual components—or in the case of synthetic biology, building new network architectures by creating DNA circuit—it is critical to control multiple genes simultaneously under tightly regulated or inducible expression. In the case of network construction in Saccharomyces cerevisiae, there has been a lack of both suitable, well-characterized parts (promoters and regulators) as well as a standardized platform for DNA assembly and delivery of gene circuits. Here, we present a framework for building gene circuits as well as a set of fully characterized DNA parts for use in Saccharomyces cerevisiae. The entire procedure of building a gene circuit from more than 10 basic parts took less than 5 days with only a workload of 1-3 hours per day. A diverse promoter collection comprising five different types was generated: constitutive, yeast native inducible, synthetic inducible, synthetic promoters regulated by activators, and synthetic promoters regulated by repressors. Altogether, the range of promoters span 2-fold to 105-fold expression above the background, the new inducible systems allow 11-fold change in expression, and the activators/repressors show a maximum 35-fold and 45-fold change of expression. This study demonstrates the feasibility for the quick and easy construction of gene circuits for delivery into S. cerevisiae and the utility of a fully characterized set of diverse promoters, activators, and repressors. This assembly system combined with DNA parts will be useful for constructing large-scale gene circuit libraries with reliable gene expression and for designing logic operations for a complex network in S. cerevisiae. Moreover, we anticipate that our system will allow for the controlled study of multi-step pathways by enabling manipulation of single protein expression.