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Abstract

System for producing a nucleic acid construct of interest, said system comprising:

a set of n entry DNAs numbered 1 to n, n being an integer of at least 2,each of said n entry DNAs comprising in this order:(i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;(ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and(iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site; the cleavage sites of the type IIs restriction endonuclease recognition sites of item (iii) of entry DNAs 1 to n−1 are complementary to the cleavage sites of the type IIs restriction endonuclease recognition sites of item (i) of entry DNAs 2 to n, respectively;the cleavage site of the type IIs restriction endonuclease recognition site of item (iii) of entry DNA n is complementary to the cleavage site of the type IIs restriction endonuclease recognition site of item (i) of entry DNA 1 for allowing annealing of complementary single-stranded overhangs formed by restriction at recognition site (i) of entry DNA 1 and at recognition site (iii) of entry DNA n;said system further comprising a destination vector comprising in this order:(I) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;(II) a vector backbone preferably comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III);(III) a further cleavage site of a type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site, and(IV) optionally, an insert between the recognition sites of item (III) and item (I);said cleavage sites of items (I) and (III) being different and non-complementary, said recognition sites of items (I) and (III) being preferably recognitions sites of the same endonuclease.

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First Claim