Abstract: A method for producing a ligated DNA formed by ligating DNA fragments is disclosed. The method includes (a1) preparing (1) a first vector containing structure 5?-R1-D(i)-R2-M1-R2?-D(ii)-R1?-3? and (2) a second vector containing structure 5?-R1-D(iii)-R2-M2-R2?-D(iv)-R1?-3?, which each contain recognition sequences of restriction enzymes R1, R1?, R2, and R2; selectable marker genes M1 and M2 different from each other; and DNA fragments for ligation D(i) to D(iv); (b1) treating the first vector with first restriction enzyme and second restriction enzyme to obtain a first vector fragment composed of structure 5?-D(i)-R2-M1-R2?-D(ii)-3; (c1) treating the second vector with third restriction enzyme and fourth restriction enzyme to obtain a second vector fragment with removed structure 5?-R2-M2-R2?-3?; and (d1) ligating the first vector fragment obtained in b1 and the second vector fragment obtained in cl by a ligation reaction to generate a third vector containing structure (3) 5?-R1-D(i)1-R2-M1-R2?-D(ii)1-R1?-3?.