Current gene synthesis methods allow the generation of long segments of dsDNA. We show that these techniques can be used to create synthetic regulatory elements and describe a method for the creation of completely defined, synthetic variants of the PHO5 promoter from the budding yeast Saccharomyces cerevisae. 128 promoters were assembled by high-temperature ligation, cloned into plasmids by isothermal assembly, maintained in E. coli, and consequently transformed into yeast by homologous recombination. Synthesis errors occurred at frequencies comparable to, or lower than those achieved with current gene synthesis methods. The promoter synthesis method reported here is robust, fast, and readily accessible. Synthetically engineered promoter libraries will be useful tools for dissecting the intricacies of promoter input-output functions, and may serve as tunable components for synthetic genetic networks.