Duggal, AryaanBackground: Herpes Simplex Virus 1 (HSV-1) is a linear dsDNA virus typically associated with ulcer development in the mouth and genital mucous membranes. Often overlooked, is the versatile DNA structure that enables HSV-1 to serve as a platform to study viral pathogenesis. HSV-1 also possesses an inherent oncolytic quality, allowing it to function as a selective therapeutic that can be programmed to target malignant cells while inducing a strong host immune response. Methods: Various endonucleases were used to isolate the genome into 11 smaller fragments. To study the effects on viral pathogenicity and oncolytic properties, we synthesized and inserted novel sequences, with a YCpBAC sequence to allow for selection and transformation into yeast spheroplasts for TAR cloning in vitro. Fragments containing Casp2 coding regions were also tested for viability, by running quantification assays and infection tests on cell lineages. Results: Isolated band size was correlated with theoretical values, indicating accurate construction of all HSV-1 fragments. After screening on -His and -Ura plates, a 200µL aliquot of 40ng/µL TAR2 sample was determined to yield a satisfactory 85% success rate in yeast assembly. The ideal quantity for viral replication in Vero E6 cells was determined to be around 1.0×10^7 cells/cm^2 providing 2.5ml of 100x viral solution. Conclusions: Successful reassembly of HSV-1 demonstrates the feasibility of using HSV-1 as a therapeutic vehicle, useful in treating cancers and understanding neurological diseases. These findings provide a strong foundation for subsequent modifications of HSV-1, which will focus on its effects on a variety of cancer cell lines.en-USsynthetic biologyoncologycancer biologyvirologypathogen-host interactionsOther