Background & Objective: Gene therapy and administration of recombinant protein are common approach in treatment of genetic disorders. But many obstacles including frequent administration of desired gene, random integration into the host genome and low expression of protein encourage scientists to design an episome which remains in high copy number in eukaryotic cells and produces desired protein in suitable manner by viral proteins. The aim of this study is designing and construction of a plasmid containing mutated large T antigen of SV40 to develop a safe vector with high replication.

  Methods: Suitable mutant for creating large T antigen was analyzed by MODELLER software and finally appropriate structures were selected. Target mutation was created in the nucleotide sequence of large T antigen by PCR method. Mutated large T antigen gene was cloned in to the IRES2-EGFP. All clones were analyzed by PCR, restriction analysis and sequencing. HEK293 and CHO cell lines were transfected by final construct and transfected cells were observed by fluorescent microscope for 40 days. Plasmid and genomic DNA were extracted from remained cells and overlap PCRs performed on them to confirm their circularity.

  Result: This plasmid, containing a mutated large T antigen of SV40, can be replicated in eukaryotic cells and then can be used in gene therapy and recombinant protein production with high safety.�

  Conclusion: The results of PCR, restriction analysis and sequencing confirm the authenticity of construct. The transfection of HEK293 and CHO cell lines showed replication of constructed plasmid in them.