This self-amplifying mRNA encodes four nonstructural proteins (nsP1–nsP4) derived from the positive-sense RNA alphavirus, Venezuelan equine encephalitis virus (VEEV), along with Oplophorus luciferase (OLuc) fused to enhanced green fluorescent protein (EGFP) via a T2A self-cleaving peptide. This bicistronic design enables independent translation of both reporter proteins from a single transcript.

OLuc is an engineered luciferase derived from the 19 kDa catalytic subunit of Oplophorus gracilirostris, a deep-sea shrimp. It exhibits approximately 150-fold greater sensitivity compared to traditional firefly luciferase and uses furimazine as the substrate for bioluminescence detection.

• 5′ capped using the vaccinia capping enzyme system with 2′-O-methyltransferase to generate a Cap 1 structure
• Substituted with Pseudo-UTP
• Partially substituted with 5mCTP
• Poly(A) tail: 180 nucleotides