Ligation-independent cloning using In-Fusion (Clontech). (A) The reaction mixture contains a vector of choice linearized by a restriction cut, a PCR product generated with primers containing 15-bp 5′ ends homologous to the ends of the linear vector (gene), and the proprietary In-Fusion enzyme. (B) The enzyme catalyzes the alignment and strand displacement of the homologous ends of the PCR product with the vector, whereas 3′-exonuclease activity removes single-stranded regions. (C) The nicks are sealed after transformation of E. coli. (Reproduced from D'Arpa 2009.)