Use of a Golden Gate plasmid set enabling scarless MoClo-compatible transcription unit assembly

Golden Gate cloning has become a powerful and widely used DNA assembly method. Its modular nature and the reusability of standardized parts allow rapid construction of transcription units and multi-gene constructs. Importantly, its modular structure makes it compatible with laboratory automation, allowing for systematic and highly complex DNA assembly. Golden Gate cloning relies on Type IIS enzymes that cleave an adjacent undefined sequence motif at a defined distance from the directed enzyme recognition motif. This feature has been used to define hierarchical Golden Gate assembly standards with defined overhangs ("fusion sites") for defined part libraries. The simplest Golden Gate standard would consist of three part libraries, namely promoter, coding and terminator sequences, respectively. Each library would have defined fusion sites, allowing a hierarchical Golden Gate assembly to generate transcription units. Typically, Type IIS enzymes are used, which generate four nucleotide overhangs. This results in small scar sequences in hierarchical DNA assemblies, which can affect the functionality of transcription units. However, there are enzymes that generate three nucleotide overhangs, such as SapI. Here we provide a step-by-step protocol on how to use SapI to assemble transcription units using the start and stop codon for scarless transcription unit assembly. The protocol also provides guidance on how to perform multi-gene Golden Gate assemblies with the resulting transcription units using the Modular Cloning standard. The transcription units expressing fluorophores are used as an example.