Fig. (1). Experimental overview.

The inserted DNA fragments were designed to contain introns with or without mutations and the adjacent exons. The primers used to amplify the fragments in PCRs included 20-bp sequences that overlapped with the terminal sequences of the digested plasmid DNA. An In-Fusion reaction was used to ligate each DNA fragment to an exon/intron cassette-trapping plasmid containing the Renilla luciferase gene and HaloTag gene (pFN21A-RL). After the construct was transfected into mammalian cells, successful splicing produced HaloTag-luciferase fusion proteins and high levels of luciferase activity, whereas aberrant splicing or no splicing did not.