The 579-nucleotide 5 untranslated region (5UTR) of the Rhopalosiphum padi virus (RhPV) possesses a cross-kingdom internal ribosome entry site (IRES) activity that functions in insect, mammalian, and plant-derived in vitro translation systems, and six TAAG motifs within the DNA fragment encoding the RhPV 5UTR were previously found to confer the RhPV 5UTR with late promoter activity in baculovirus. In the present study, various truncated RhPV 5UTR sequences were produced, and among them, a fragment of 110 bp ranging from nucleotides 309 to 418 was identified to be the shortest fragment responsible for the late promoter activity in baculovirus infected Sf21 cells. This 110 bp fragment contains a TAAG tandem repeat that retains more than 60% of the late promoter activity of the full length RhPV 5UTR sequence. Further, IRES activity remained unchanged in all truncated RhPV 5UTR constructs. Taken together, this novel 110 bp fragment having late promoter activity in baculovirus as well as IRES activity in mammalian cell, renders it a useful tool for the development of a shuttle bi-cistronic baculovirus gene expression and/or delivery vector.