Abstract: Disclosed are methods and constructs for engineering circular RNA Disclosed is a vector for making circular RNA, said vector comprising the following elements operably connected to each other and arranged in the following sequence: a) a 5? homology arm, b) a 3? group I intron fragment containing a 3? splice site dinucleotide, c) an optional 5? spacer sequence, d) a protein coding or noncoding region, e) an optional 3? spacer sequence, f) a 5? Group I intron fragment containing a 5? splice site dinucleotide, and g) a 3? homology arm. This vector allows production of a circular RNA that is translatable or biologically active inside eukaryotic cells. In one embodiment, the vector can comprise the 5? spacer sequence, but not the 3? spacer sequence. In yet another embodiment, the vector can also comprise the 3? spacer sequence, but not the 5? spacer sequence.

Abstract: This invention relates to the fields of mRNA vaccines, mRNA therapy, and gene therapy and specifically to the use of gene expression vectors or PCR amplicons containing various 5?UTR sequences followed by coding sequences for in vitro and in vivo production of mRNA or proteins of interest.