Abstract
Existing synthetic biology toolkits have expanded part libraries and assembly capabilities. However, new users may still encounter ambiguity in construct definitions, hierarchical transitions, genomic integration routes, and quantitative testing procedures. Here, we present Polaris, a Golden Gate toolkit for Escherichia coli and Saccharomyces cerevisiae. Polaris defines a shared three-level hierarchy. Level 1 stores reusable parts, Level 2 generates transcription units and other intermediate constructs, and Level 3 supports multigene assembly and switching between testing and integration backbones. The toolkit distinguishes part interfaces from transcription unit interfaces, uses BpiI and BsaI in successive assembly stages, and provides parallel ccdB and mScarletI3 screening options. Integration modules and quantitative characterization frameworks were implemented in both hosts and demonstrated using representative genetic parts and activation and repression systems. A companion digital platform further encodes the Level 1 to Level 3 rules for assembly planning. Polaris connects hierarchical construction, stable genetic implementation, and quantitative testing within a common execution framework, providing a practical basis for data accumulation, part standardization, and future automation.