Compositions and production of nicked closed-ended DNA vectors

A method of producing a closed-ended DNA vector having one or more gaps (neDNA) comprising:

• providing a double-stranded DNA construct comprising an expression cassette, wherein the expression cassette comprises a promoter linked to a transgene, wherein a 3′ end and a 5′ end of said double-stranded DNA construct each comprise an overhang sequence;
• providing a first inverted terminal repeat (ITR) comprising an overhang sequence that comprises a complement to at least a portion of the overhang sequence of the 5′ end of the double-stranded DNA construct, wherein the first ITR is closed-ended (5′ ITR);
• providing a second ITR comprising an overhang sequence that comprises a complement to at least a portion of the overhang sequence of the 3′ end of the double-stranded DNA construct, wherein the second ITR is closed-ended (3′ ITR);
• contacting said double-stranded DNA construct comprising the expression cassette with the first ITR, the second ITR, and a ligase, wherein ligation of the first ITR and the second ITR with the double-stranded DNA construct comprising the expression cassette produces (i) the closed-ended DNA vector having one or more gaps in a sense or antisense strand 5′ upstream of the expression cassette, or 3′ downstream of the expression cassette, or (ii) the closed-ended DNA vector having one or more gaps in a sense or antisense strand both 5′ upstream and 3′ downstream of the expression cassette.