Fig. 1: A modular cloning platform for circRNA enables rapid design–build–test cycles.
From: Engineering circular RNA for enhanced protein production
Schematic describing the modular cloning platform used to create template plasmids for circRNA synthesis. Parts 1–6 corresponding to the upstream intron and 5′ UTR, IRES, N-terminal (N′) tag, coding sequence (CDS), C-terminal (C′) tag and 3′ UTR and the downstream intron were individually cloned into part plasmids via Golden Gate reactions (Supplementary Fig. 1). Part plasmids and the circRNA backbone were then combined in a second Golden Gate reaction to create a circRNA plasmid. The circRNA backbone contains a CAG promoter enabling circRNA transcription after transient transfection in cellulo, a T7 promoter enabling IVT, homology sequences that assist with RNA circularization, low-structure regions that facilitate RNaseR processivity and a bacterially expressed GFP dropout sequence to negatively select for incorrect assemblies. If a CDS without N′ or C′ tags was used, parts 3–5 were replaced with a single part. PCR products from circRNA plasmids were subsequently used as templates for IVT to synthesize RNA. Lastly, RNaseR cleanup was performed to digest linear RNAs and isolate circRNA. DS, downstream.
