The Gibson Assembly® Ultra reaction occurs in two steps using the addition of two master mixes in two sequential steps. In the first step, the GA Ultra Master Mix A (2X) creates single-strand DNA 5’ overhangs by chewing back DNA from the 3’ end. This reaction is cooled under conditions that allow for annealing of complementary overlap regions. Next, the Gibson Assembly® Ultra Master Mix B (2X) is added. During this step, nucleotides are incorporated into the construct to fill in the gaps in the annealed DNA fragments and nicks are sealed to create a contiguous DNA construct.