I am working with the pETDuet and pRSFDuet plasmids to express RNAs under control of the T7 promoter. On these plasmids the antibiotic resistance genes (Amp for pET, and Kan for pRSF) are downstream of the T7 promoter site where I cloned in my genes. The Amp and Kan genes are transcribed in the opposite orientation of the T7 promoter and since these genes are downstream from where I inserted my genes, readthrough transcription from the resistance genes would result in a sequence complementary to the RNA sequence I wish to produce. My question is: do the antibiotic resistance genes in these plasmids have a built in transcription termination mechanism?

I have used the ARNold tool: http://rssf.i2bc.paris-saclay.fr/toolbox/arnold/index.php to look for Rho-independent terminators at the 3' end of the antibiotic resistance genes and in the region downstream. There was one hit for the pETDuet plasmid but nothing for the pRSFDuet plasmid. There were no hits for the pCDFDuet or pCOLADuet plasmids either. My sense is hairpin terminators were not specifically incorporated to stop transcription downstream of the antibiotic resistance genes. The other possibility is the antibiotic resistance genes terminate in a Rho-dependent manner, is this known to occur for the Amp and Kan genes?


Generally they do, however this is often doesn't completely stop transcription run-through.

Note that in a T-7 reaction there shouldn't be any transcription of the Amp or Kan genes as these are run off promoters that the T7 phage RNA polymerase won't be able to transcribe off as it is extremely specific for the T7 promoter.

The solution if you are really worried is to linearize your plasmid immediately downstream of your terminator but before the AMPR gene using a restriction enzyme with a single cut. This will also give you a specific size sequence being transcribed, which is very useful if you are determining copy number in say a qPCR.


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