Cell-free method of producing synthetic circular nucleic acid

A method of amplifying a synthetic circular nucleic acid, comprising:

• a) providing a circular nucleic acid template comprising:
• (i) a backbone, wherein the backbone comprises one or more endonuclease cut sites, and
• (ii) an insert sequence, wherein the insert sequence comprises one or more endonuclease cut sites on its 5′ and 3′ terminal ends;
• b) amplifying the circular nucleic acid template by rolling circle amplification, thereby producing an amplification product;
• c) contacting the amplification product with a first endonuclease under conditions for digestion, thereby producing a first digestion product;
• d) adding the first digestion product to a ligation reaction mixture comprising a ligase enzyme, wherein the first digestion product is added to the ligation reaction mixture gradually, continuously, or intermittently over time;
• e) incubating the ligation reaction mixture to produce a circular ligation product, wherein the circular ligation product is at least partially supercoiled;
• f) optionally contacting the circular ligation product with a topoisomerase enzyme under conditions suitable to promote supercoiling;
• g) contacting the circular ligation product with a first exonuclease that digests, linear nucleic acid, and open circle nucleic acid, thereby producing a final reaction product comprising synthetic circular nucleic acid that is substantially free of linear nucleic acid;
• and
• h) purifying the final reaction product by a purification method.