Abstract: DNA molecules for making a circular RNA are provided. The DNA molecule may include elements operably connected and arranged, from a 5? to 3? direction, in the following order: (a) an intron fragment that includes a full-length intron; (b) an E2 fragment which includes a downstream exon of the full-length intron; and (c) an E1 fragment which includes an upstream exon of the full-length intron. 3? end of the E1 fragment is configured to produce a hydroxyl group in an in vitro transcription reaction; the hydroxyl group is capable of initiating splicing in a one-step transesterification reaction at a splice site between RNA fragments transcribed from the intron fragment and the E2 fragment in a linear RNA that is produced from the DNA molecule in the in vitro transcription reaction, such that the linear RNA is configured to self-circularize to produce the circular RNA.