In last decades great efforts have been devoted to the study of development of recombinant peptide based vectors that consist of biological motifs with potential applications in gene therapy. Recombinant Biomimetic Chimeric Vectors (rBCVs) are biopolymeric nanocarriers that are designed to mimic viral features to overcome the cellular obstacles in gene transferring pathway into cell nucleus. In this research, we designed and genetically engineered three novel rBCVs with similar sequences that differed in motifs arrangement and motif abundance: MPG-2H1, 2TMPG-2H1 and 2RMPG-2H1. The MPG as a famous amphipathic cell penetrating peptide is the main segment of these constructs which was studied for the first time in association with truncated histone H1 DNA condensing motif. Through the performance of several physicochemical and biological assays, the rBCVs were remarkably examined regarding transfection efficiency. The main objective of this study is focused on the importance of motif design in transfection efficiency of rBCVs on one hand, and the assessment of correlation between structural features and functionality of motifs on the other hand. The results revealed that all three kinds of rBCVs/pDNA nanoparticles with average sizes of 200 nm could overwhelm the cellular obstacles associated with gene transfer, and lead to efficient gene delivery. Furthermore, no significant toxicity was perceived and efficient endosome disruptive activity was obtained. It is noteworthy to say among three mentioned constructs 2RMPG-2H1 showed the highest transfection efficiency. Overall the peptide based vectors hold great promise as a nontoxic and effective gene carrier in vitro and in vivo, besides the rational design possibility as the most vital advantages over the other non-viral gene delivery vectors.