Compositions and methods for high fidelity assembly of nucleic acids

A method of producing a target nucleic acid having a predefined sequence, the method comprising:

• providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends of each of the plurality of blunt-end double-stranded nucleic acid fragments;
• producing a plurality of cohesive-end double-stranded nucleic acid fragments that together comprises the target nucleic acid sequence via enzymatic digestion of the plurality of blunt-end double-stranded nucleic acid fragments, wherein the plurality of cohesive-end double-stranded nucleic acid fragments each have two different and non-complementary overhangs;
• ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase, wherein a first overhang of a first cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a second overhang of a second cohesive-end double-stranded nucleic acid fragment;
• and
• forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid having a predefined sequence.