The formation of linear RNA and circRNA from pre-RNA is a competitive process. The factors that determine which type of RNA molecule is formed include the sequence of the pre-mRNA, the presence of regulatory elements, and the activity of RNA-binding proteins. In this study, multiple circRNAs can be formed by introducing the luc expression vector or in virto transcribed luc RNAs into three cell lines, HepG2, BmN, CIK cells, which are models widely used for studying hepatocellular carcinoma,silkworm molecular biology, and Ctenopharyngodon idellus-pathogens interaction, respectively. The existence of luc-derived circRNAs was clarified by reverse transcription-PCR, Sanger sequencing, and reverse transcription-rolling circle amplification. Sequence analysis of the circRNAs junction sites showed that the back splicing-like(BSL) sites of these luc-circRNAs were different from the canonical splice sites.The mutations at BSL sites can significantly reduce the expression levels of luc-derived circRNAs, and increase the expression levels of luc mRNA in cells. Furthermore, luciferase activity was significantly increased with BSL site mutations transfected cells. In addition, the efficiency of circRNA formation was reduced and corresponding RNAs was increased in cells infected with recombinant baculoviruses with BSL site mutations. Furthermore, silencing of circRNA formation-related protein genes significantly reduced the expression efficiency of luc-derived circRNAs and significantly increased the expression level of luc.These studies will provide valuable insights into the regulation of BSL-mediated gene expression and contribute to the development of more efficient and targeted gene expression systems.~2