Large DNA constructs (>10 kb), including small genomes and artificial chromosomes, are invaluable tools for genetic engineering and vaccine development. However, the manufacture of these constructs is laborious. To address this problem, we applied new design insights and modified protocols to Golden Gate assembly. While this methodology is routinely used to assemble 5-10 DNA parts in one-step, we found that optimized assembly permitted >50 DNA fragments to be faithfully assembled in a single reaction. We applied these insights to genome construction, carrying out rapid assembly of the 40 kb T7 bacteriophage genome from 52 parts and recovering infectious phage particles after cellular transformation. The new Golden Gate assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.
