Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant morbidity and mortality in tropical and sub-tropical areas worldwide. Despite an urgent need, safe and effective antiviral therapeutics and vaccines are not available. This is partly attributable to significant gaps in our understanding of the exact features of the viral genome and its encoded proteins that are critical to the viral replication cycle. Here we subjected an infectious DENV genome to unbiased insertional mutagenesis and employed next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in Huh-7.5 hepatic cell culture and following passage of the resulting mutant library onto Vero and C6/36 cell lines. This revealed that broad regions within capsid, NS1 and the 3’UTR were most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high resolution imaging of its localization to the surface and interior of viral replication vesicles. Additionally, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that enabled live cell imaging of NS1 localization and traffic in infected cells. This analysis revealed that large juxtanuclear NS1 foci are relatively static while smaller NS1 foci frequently display rapid bi-directional traffic. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and application and in future antiviral strategies.