To optimize the efficiency of a Minimalistic Immunogenically Defined Gene Expression (MIDGE) vector, peptides containing proven (SV40 T-antigen and bovine herpesvirus-1 VP8) or putative (herpes simplex virus-1 VP22) nuclear localization signals (NLSs) were linked to a MIDGE vector encoding a truncated, secreted form of BHV-1 glycoprotein D (tgD) (MIDGE-tgD). Conjugation of an NLS to the MIDGE-tgD vector improved the tgD expression in vitro and the humoral and cellular immune responses induced in mice in vivo. The NLS from BHV-1 VP8 was most efficient at enhancing the tgD production and tgD-specific immune responses when conjugated to the MIDGE-tgD vector.