  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.
