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To identify appropriate ligation sites, by selecting several potential ligation sites with fewer mutations around two short linker regions, their ligation efficiencies and the functions of the ligated ER\u201060s were examined experimentally. To evaluate the dependence of ligation efficiencies on the ligation sites computationally, steric hinderances around the sites for the ligation were calculated through molecular dynamics simulations. Utilizing the steric hindrance, a site\u2010dependent ligation potential index was introduced as reproducing the experimental ligation efficiency. Referring to this index, the reconstruction of ER\u201060 was succeeded by the ligation of the three domains for the first time. In addition, the new ligation potential index well\u2010worked for application to other domain ligations. 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