Methods and compositions relating to covalently closed nucleic acids

A method for assembly of a covalently closed double stranded nucleic acid, comprising:

• (a) providing a double stranded nucleic acid;
• (b) amplifying the double stranded nucleic acid using a primer comprising one or more uracils to generate a double stranded nucleic acid comprising one or more uracils at a 5′ end and a 3′ end;
• (c) digesting the double stranded nucleic acid comprising one or more uracils at the 5′ end and the 3′ end using a glycosylase and a glycosylase-lyase to generate a double stranded nucleic acid comprising a loop structure at the 5′ end and the 3′ end;
• and
• (d) ligating gaps in the double stranded nucleic acid comprising the loop structure at the 5′ end and the 3′ end using a ligase to generate the covalently closed double stranded nucleic acid,
• wherein the loop structure comprises a nucleic acid sequence according to SEQ ID NO:
• 9, SEQ ID NO:
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• 19, or SEQ ID NO:
• 20.