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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
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    $\begingroup$ you can use bidirectional promoteror you may extend the spacer to ~100bp just use the 10bp spacer in tandem or better, with some reshuffling.. $\endgroup$
    – WYSIWYG
    Commented Jul 25, 2013 at 3:05
  • $\begingroup$ The spacer is a good idea, can you maybe elaborate on the reshuffling? $\endgroup$
    – bsubspore
    Commented Jul 25, 2013 at 7:40
  • $\begingroup$ just use similar sequence but not exactly the same sequence.. you can move some bases here and there.. though, not really essential to do so, it will reduce repeats.. $\endgroup$
    – WYSIWYG
    Commented Jul 25, 2013 at 22:20

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If you want to express them in an operon, make sure you have an RBS at the 5' end of each coding sequence. If you place one 10bp downstream without it having an RBS, it will be transcribed, but not translated... If you want more help, paste the DNA code of each as a comment.

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