Gene expression in mycobacteria relies on both integrative and episomal Escherichia coli-mycobacterium shuttle vectors. However, existing vectors have limited flexibility for adding epitope tags, selectable markers, and fluorescent reporters. To overcome this, we created a modular toolkit consisting of 32 integrative and eight episomal E. coli-mycobacterium shuttle vectors. Each vector features N- or C-terminal tags: SBP-HA-S, 3×-FLAG, GFP, and mCherry. The integrative vectors cont