I want to clone two fluorescent proteins both driven by different promoters in a plasmid to be used to transform B. subtilis at the amyE locus. For this I want to use plasmid pSG1729 (Lewis and Marston, 1999), because this plasmid can be used for homologous recombination at this specific locus.
Now the problem is, because of a few reasons (i.e., the locations of the restriction sites, keeping the spc resistance gene and deleting the current GFP gene), if I want to clone two genes into this plasmid, they will be very close to each other (~10bp). Will this be a problem either technically or biologically after transformation? I don't think that possible read-through will be bad, as I want one of the fluorescent proteins (pHluorin) to be continuously expressed. Maybe an option would be to clone the two genes in opposite directions?
This is the current genotype of the plasmid:
Ori amyE5' currentGFP Spc amyE3' bla