Closed linear DNA production

A cell-free method of producing closed linear deoxyribonucleic acid (DNA) molecules, the method comprising:

• (a) contacting a DNA template comprising a linear, double stranded DNA molecule covalently closed at each of its ends by a portion of a protelomerase recognition sequence and comprising at least one stem loop motif with a strand-displacing polymerase under conditions promoting amplification of said DNA template in the presence of at least one primer which is capable of hybridizing specifically to a primer binding site within said stem loop motif;
• and
• (b) producing the closed linear DNA molecules by contacting DNA produced in step (a) with at least one protelomerase under conditions promoting production of the closed linear DNA molecules;
• wherein a sequence of the stem loop motif comprises a central section and two flanking sequences, and wherein the central section comprises the primer binding site, wherein the sequence of the stem loop motif is capable of forming a stem loop secondary structure under conditions promoting formation of a secondary structure,
• wherein the central section of the sequence of the stem loop motif is a single stranded DNA region in the stem loop secondary structure, and
• wherein the sequence of the stem loop motif is different from the portion of the protelomerase recognition sequence of the DNA template, and the stem loop motif and the portion of the protelomerase recognition sequence are located in different parts of the DNA template.