Reverse genetics systems are a critical tool in combating emerging viruses by enabling a better understanding of the genetic mechanisms by which they cause disease. Traditional cloning approaches are fraught with difficulties due to the bacterial toxicity of many viral sequences. Many attempts have been made to develop a simplified workflow to produce a simple-to-use infectious clone without the concerns of host toxicity; however, none offers the advantages of bacterial cloning without the various disadvantages. This paper demonstrates a novel in vitro workflow that leverages gene synthesis and replication cycle reaction—an innovative technology that reconstitutes the bacterial replication machinery in a tube—to develop a supercoiled infectious clone plasmid that is easy to rescue. Using this workflow, we developed two infectious clones, one of a low passage dengue virus serotype 2 isolate (PUO-218) and the Washington strain of SARS-CoV-2, which behaved similarly to their respective parental viruses. Furthermore, we generated a medically relevant mutant of SARS-CoV-2, Spike D614G. These results indicate that this novel in vitro workflow is a viable method to generate and manipulate infectious clones, specifically for viruses that are notoriously difficult for traditional bacterial-based cloning methods. This work represents a paradigm shift in producing infectious clones and lowers the barrier of entry for scientists to develop such tools.