Provided is a gene expression cassette for stably and highly producing a protein of interest. The gene expression cassette has a structure in which a DNA construct (X) containing a gene of interest and a poly A addition sequence is sandwiched between a promoter (P) and an enhancer (P′), the gene expression cassette further including transposon sequences (T) upstream of the promoter (P) and downstream of the enhancer (P′). Further, in the gene expression cassette, when a nuclear matrix binding sequence (M) is appropriately arranged upstream of a replication initiation sequence (S) in combination with the transposon sequence (T), the protein of interest can be more effectively produced stably and in a large amount. For example, HRG, PD-1, EMMPRIN, NPTNβ, EMB, RAGE, MCAM, ALCAM, ErbB2, and an antibody can each be produced stably and in a large amount.
