The emergence of multidrug-resistant pathogens has motivated natural product research to inform the development of new antimicrobial agents. Glycocin F (GccF) is a diglycosylated 43-amino acid bacteriocin secreted by Lactobacillus plantarum KW30. It displays a moderate phylogenetic target range that includes vancomycin-resistant strains of Enterococcus species and appears to have a novel bacteriostatic mechanism, rapidly inhibiting growth of the most susceptible bacterial strains at pM concentrations. Experimental verification of the predicted role(s) of gcc cluster genes in GccF biosynthesis has been hampered by the inability to produce soluble recombinant Gcc proteins. Here we report the development of pRV610gcc, an easily modifiable 11.2 kbp plasmid that enables the production of GccF in L. plantarum NC8. Gcc gene expression relies on native promoters in the cloned cluster, and NC8 pRV610gcc produces mature GccF at levels similar to KW30. Key findings are that: the glycosyltransferase glycosylates both serine and cysteine at either position in the sequence, but glycosylation of the loop serine is both sequence and spatially specific; glycosylation of the peptide scaffold is not required for export and subsequent disulfide bond formation; neither of the putative thioredoxin proteins is essential for peptide maturation; removal of the entire putative response regulator GccE decreases GccF production less than removal of the LytTR domain alone. Using this system, we have verified the functions of most of the gcc genes and have advanced our understanding of the roles of GccF structure in its maturation and antibacterial activity. IMPORTANCE The entire 7-gene cluster for the diglycosylated bacteriocin glycocin F (GccF), including the natural promoters responsible for gcc gene expression, has been ligated into the E. coli-LAB shuttle vector pRV610 to produce the easily modifiable 11.2 kbp plasmid pRV610gcc for the efficient production of glycocin F analogues. In contrast to the refactoring approach, chemical synthesis, or chemoenzymatic synthesis, all of which have been successfully used to probe glycocin structure and function, this plasmid can also be used to probe in vivo the evolutionary constraints on glycocin scaffolds and their processing by the maturation pathway machinery, thus increasing understanding of the enzymes involved, the order in which they act and how they are regulated.