text: Although Golden Gate Cloning speeds up multisegment cloning, careful design of donor and recipient plasmids is required. Scientists at New England Biolabs have successfully demonstrated the assembly of 35 fragments via a single-tube Golden Gate Assembly reaction. Critical to this method of assembly, the vector backbone of the destination plasmid and all the assembly fragments are flanked by Type IIS restriction enzyme recognition sites, as this subtype of restriction enzymes cut downstream from their recognition sites. After cutting, each assembly active piece of DNA has unique overhangs that anneal to the next fragment of DNA in the planned assembly and become ligated, building the assembly. While it is also possible for an overhang to anneal back to its original complementary overhang associated with the upstream recognition site and become ligated, re-forming the original sequence, this will be susceptible to further cutting throughout the assembly reaction.