Assembly of three DNA fragments using NE-LIC coupled with in vivo circularization.

(A) Schematic diagram of the three DNA fragments assembly. (B) Three fragments assembly using both 3′-end (5′-end digestion using Nb.BbvCI) (left) and 5′-end single-stranded (3′-end digestion using Nt.BspQI) (right) annealing. DNA gel electrophoresis verified the assembly results (B-middle).