Baculovirus Expression Vector System is as an important tool for molecular biology and widely used in insect cells and mammalian cells. Development of a shuttle baculovirus expression vector which can function in both the mammalian cell as well as in insect cell would have a significant advantanges. Previous studies have demonstrated that the internal ribosome entry site (IRES) derived from 5’ untranslated region (5’ UTR) of Rhopalosiphum padi virus (RhPV) can mediate cap-independent translation in mammals, plants and insect cells by in vitro translation system. Moreover, earlier studies in our laboratory revealed the presence of cryptic promoter in RhPV 5’ UTR containing IRES in baculovirus infected Sf21 cells. Series of deletion experiments were conducted in six baculovirus late promoter conservative motifs (TAAG) in the cDNA of RhPV 5’ IRES to determine the specific motif which provide cryptic promoter activity of RhPV 5’ IRES. Two tandem repeated TAAG motifs within 309 ~ 418 nts of the 579 nts was responsible for critical promoter activity in baculovirus infected Sf21 cells and the sequence segment was named RP110. Furthermore, RP110 was implied to deletions and point mutations in its TAAG motifs to investigate the promising critical cryptic promoter region. TAAG motif deletion results implied that both the motifs were necessary for the promoter activity of RP110 and the point mutation experiment indicated the first mutation on TAAG of RP110 to have promising promoter activity than the second mutation. However, an interesting observation is the presence of promoter activity in baculovirus infected Sf21 cells and partial IRES acitivity in CHO cells when motifs are subjected to deletions. Thus, chimeric sequence was formed by fusing RP110 with cDNA derived 5’UTR of enterovirus 71 to generate an artificial sequence that can function as a promoter in baculovirus infected Sf21 cells and also provide IRES activity in mammalian cells. In conclusion, the chimeric sequence will facilitate the development of a dual-baculovirus shuttle expression vector that can express genes in insect cells as well as mammalian cells.