Objective: The invention of induced pluripotent stem (iPS) cells enables for the first time the derivation of enough numbers of patient-specific stem cells and holds promise for cell-based therapies. Recent studies have revealed the potential of iPS cell generation combined with gene therapy for treatment of different disease in human. In this study we designed and constructed a nonviral plasmid vector as a gene transfer vehicle. This vector is tissue-specific and its integration in the genome is site specific. We expect to have persistent and acceptable level of b-globin expression as transgene per genomic copy from the vector. This vector will be used for genetic manipulation of iPS cells derived from patients with b-thalassemia major or sickle cell anemia with potential for clinical application.Materials and Methods: In the first step, an appropriate nonviral plasmid vector was designed and constructed by genetic engineering approaches. This vector comprises two main parts, a bacterial backbone for efficient amplification in E. coli cells and one expression cassette containing b-globin gene enhancers, promoter and b-globin gene sequence along with its 3´ & 5´ UTRs and one eukaryotic antibiotic resistance gene against puromycin. By adding a specific phage integrase site in the structure of this vector, integration occurs in specific sites in the genome (Site Specific Recombination; SSR). Also by designing of two loxP sites flanking of bacterial backbone and puromycin ORF it is possible to delete these sequences by Cre recombinase after integration of vector in the genome of target cells. These characteristics reduce possible host immune responses to the vector and potential genotoxicity. The structure of the vector was confirmed through several steps of digestion experiments and sequence analysis. Functional assessment of plasmid vector, which is named pHBB, was achieved by transient transfection in to hematopoietic cell lines, which are b-globin promoter specific and able to express b-globin gene.Results: The recombinant expression vector containing b-globin gene was successfully constructed and confirmed. To study of its activity, the vector lipofected in to suitable hematopoietic cell lines transiently and the expression of the gene under control of b-globin promoter was detected.Conclusion: This nonviral plasmid vector is applicable to correct the defects of b-globin gene by adding a functional copy of b-globin gene in stem cells derived from the patients who have the b-thalassemia major or sickle cell anemia (gene addition method) using site specific recombination. The production about 10% or more hemoglobin in RBCs of these patients compare to normal people can help to treat these disease.