Construction of the FLK-1 DNA minigene vaccine. (A) Minigenes encoding the HIVtat translocation peptide, a spacer (AAA), and murine FLK-1 H-2D^b– and K^b-restricted epitopes (pHI-Db or pHI-Kb, respectively), were assembled by polymerase chain reaction (PCR) with overlapping oligonucleotides as templates. D^b-restricted epitopes include FLK₉₄: RVVGNDTGA; FLK₄₀₀: VILTNPISM; FLK₁₂₁₀: FHYDNTAGI. K^b-restricted epitopes include FLK₅₄: RGQRDLDWL; FLK₇₇₁: VIAMFFWLL; FLK₁₁₂₉: TTPEMYQTM. The PCR fragments generated were cloned into a pCMV vector at C-terminal of ER signal peptide (endoplasmic reticulum) by using BssH II and XhoI restriction sites. (B) Proteins encoded by minigenes were expressed in mammalian cells. This was indicated when 293T cells were transfected with either pHI-Db-myc or pHI-Kb-myc for 24 hours, harvested, lysed, and analyzed by Western blotting with anti-myc monoclonal antibody.