A non-viral method for inducing pluripotency and removing reprogramming factors is described. The study uses a single multiprotein expression vector containing c-Myc, Klf4, Oct4, and Sox2 linked by 2A peptides, which can reprogram both mouse and human fibroblasts. The vector is non-viral and allows for the removal of reprogramming factors once reprogramming is achieved. The resulting iPS cells show robust expression of pluripotency markers and can be differentiated in vitro and form chimeric mice in vivo. When combined with a piggyBac transposon, the system enables the generation of reprogrammed human cell lines with robust pluripotency markers. The system minimizes genome modification and allows for the complete elimination of exogenous reprogramming factors, making the iPS cells suitable for regenerative medicine, drug screening, and disease modeling. The study also demonstrates that the reprogramming cassette can be excised using Cre transfection, leading to the removal of exogenous factors and maintaining pluripotency. The non-viral system is efficient and avoids the risks associated with viral integration. The study shows that the system can be applied to human cells, generating exogenous factor-free iPS cells. The results indicate that the non-viral single-vector system is effective for reprogramming and that the generated iPS cells are genuinely pluripotent. The study also highlights the importance of avoiding exogenous reprogramming factor reactivation for clinical applications and drug screening. The system is ideal for regenerative medicine due to its ability to generate non-genetically modified human iPS cells.A non-viral method for inducing pluripotency and removing reprogramming factors is described. The study uses a single multiprotein expression vector containing c-Myc, Klf4, Oct4, and Sox2 linked by 2A peptides, which can reprogram both mouse and human fibroblasts. The vector is non-viral and allows for the removal of reprogramming factors once reprogramming is achieved. The resulting iPS cells show robust expression of pluripotency markers and can be differentiated in vitro and form chimeric mice in vivo. When combined with a piggyBac transposon, the system enables the generation of reprogrammed human cell lines with robust pluripotency markers. The system minimizes genome modification and allows for the complete elimination of exogenous reprogramming factors, making the iPS cells suitable for regenerative medicine, drug screening, and disease modeling. The study also demonstrates that the reprogramming cassette can be excised using Cre transfection, leading to the removal of exogenous factors and maintaining pluripotency. The non-viral system is efficient and avoids the risks associated with viral integration. The study shows that the system can be applied to human cells, generating exogenous factor-free iPS cells. The results indicate that the non-viral single-vector system is effective for reprogramming and that the generated iPS cells are genuinely pluripotent. The study also highlights the importance of avoiding exogenous reprogramming factor reactivation for clinical applications and drug screening. The system is ideal for regenerative medicine due to its ability to generate non-genetically modified human iPS cells.