Background: Tuberculosis remains the major infectious and contagious disease with respect to morbidity and mortality around the globe. The main etiological agent of TB is Mycobacterium tuberculosis (M. tb). The cure and/or control of this disease is getting difficult day by day especially due to emergence of drug resistant strains, HIV co-infection and unavailability of good vaccines. BCG is the only available and permitted vaccine against TB, which has variable efficacy only limited to childhood. Therefore, new, well defined, antigen specific and state of the art Molecular Biology based DNA vaccines are required.Methods: The current study is designed to develop subunit-based DNA vaccines against tuberculosis by using five different Mycobacterium specific genes, namely Rv1908c/KatG, Rv0350/DnaK, Rv0440/GroEL2, Rv0934/PstS and Rv3418c/GroES. All the selected genes were amplified through PCR and cloned into mammalian expression pVAX1 vector.Results: All the transformed constructs were confirmed through restriction digestion, colony PCR and sequence analysis. These constructs will be used for various in Vivo immunization studies and also for the challenge studies against TB in future experiments.Conclusion: DNA vaccines, alone or in combination with BCG, have enough potential to be a good therapeutic tool for TB and reduce the treatment time in future.Keywords: DNA vaccine; pVAX1; Tuberculosis; Vaccines