The pcDNA3 mammalian expressionvector uses the cytomegalovirus (CMV)promoter to express proteins cloned into anadjacent polylinker. We have modified this vector to create three different targetingconstructs: Go to Plasma Membrane(pGTM), Go to Nucleus (pGTN) and Go toNucleus, Activate Transcription (pGTNAT).pGTM expresses a protein as a fusion toamyristic acid attachment signal, which tar-gets proteins to the cell membranes. pGTNexpresses an inserted protein as a fusion toa nuclear localization sequence (NLS), tar-geting it to the nucleus. pGTNAT incorpo-rates an NLS (directing proteins to the nu-clear compartment), an acid blob (atranscriptional activation domain) andahemagglutinin epitope tag, creating a 107-amino acid fusion domain. We then clonedgreen fluorescent protein (GFP) into thethree novel vectors and pcDNA3, and wetransfected HeLa cells to test the new tar-geting constructs. Immunofluorescenceanalysis showed that the GFP protein lo-calizes to the nucleus when over-expressedin pGTN and pGTNAT, localizes to theplasma membrane and perinuclear mem-brane when in pGTM and is ubiquitousthrough the cell in pcDNA3. We anticipatethat these new vectors will prove very use-ful in future expression studies to examinethe function of particular proteins whenthey are localized to specific cellular com-partments.