A method for improving the replication of covalent closed circular plasmids is provided. [Solution] The method comprises the step of preparing a covalent closed circular
plasmid having a Pol I-dependent
origin of replication and an insert containing a structured
DNA sequence selected from the group consisting of a reverse
repeat sequence, a directional
repeat sequence, a homopolymer
repeat sequence, a eukaryotic
origin of replication, or a eukaryotic
promoter enhancer sequence, wherein the structured
DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent
origin of replication in the direction of replication. The method also comprises the step of modifying the covalent closed circular recombinant molecule so that the Pol I-dependent origin of replication is replaced with a Pol III-dependent origin of replication, thereby improving the replication of the covalent closed circular
plasmid with the resulting Pol III-dependent origin of replication. A covalent closed circular
recombinant DNA molecule without antibiotic markers is also provided.