Gordonia, a Gram-positive, filamentous bacterial genus within the phylum Actinobacteria, thrives in diverse environments and exhibits significant catabolic capabilities, capable of degrading heavy metals and hydrocarbons, making them ideal candidates as potential bioremdiators. In wastewater treatment plants (WWTPs), Gordonia spp. are advantageous for mitigating pollutants which aid in the processing of wastewater. However, excessive proliferation of Gordonia species, particularly G. amarae, in WWTPs can lead to stable foaming, posing biological, environmental, and operational challenges to treatment plants. Genetic studies of G. amarae may provide researchers with insights into their behaviour through the manipulation and transfer of genes between related species. Although, despite their importance, genetic manipulation of Gordonia species remains challenging due to limited knowledge of plasmids that replicate within them. This study focuses on plasmids capable of replicating in G. amarae, specifically a large native plasmid pBEN371, from G. pseudoamarae. Gene annotation of pBEN371 revealed the repA and oriV region which enabled the development of a shuttle vector for gene cloning in G. amarae CON44T and related species. This research has enhanced the genetic manipulation toolkit of G. amarae and in turn may facilitate the development of effective strategies for managing Gordonia mediated foaming while leveraging their bioremediation potential.