I can not find Kana promoter in the map, e.g., pet28, while AmpR promoter is always indicated. Then what is the Kana promoter sequence?
And can E. coli containing a plasmid with KanMX(TEF promoter and terminator) resist to Kanamycin?
To answer your first question, yes, E. coli transformed with KanMX should be kan-resistant. For your first question, I believe the kan gene is under the control of the native promoter. Below I quote the Novagen pET System Manual to give you some additional info:
Another difference between kanR and most of the ampR pET vectors involves the direction of transcription of the drug resistance gene. In kanR pET vectors, the kan gene is in the opposite orientation from the T7 promoter, so induction of the T7 promoter should not result in an increase in kan gene product. In contrast, in most ampR pET vectors the β-lactamase gene is located downstream and in the same orientation as the T7 promoter. All pET translation vectors have the native T7 transcription terminator (Tφ) located before the β-lactamase gene. However, this terminator is only approximately 70% effective, allowing T7 RNA polymerase read-through to produce a small amount of β-lactamase RNA in addition to the target RNA. This results in the accumulation of β-lactamase enzyme in induced cultures. Accordingly, the orientation of the β- lactamase gene has been reversed in the pET-43.1 and pET-44 vectors, so that read-through by the T7 RNA polymerase will not result in increased levels of β-lactamase gene product.
To answer your first question, yes, E. coli transformed with KanMX should be kan-resistant. For your first question, I believe the kan gene is under the control of the native promoter. Below I quote the Novagen pET System Manual to give you some additional info:
Another difference between kanR and most of the ampR pET vectors involves the direction of transcription of the drug resistance gene. In kanR pET vectors, the kan gene is in the opposite orientation from the T7 promoter, so induction of the T7 promoter should not result in an increase in kan gene product. In contrast, in most ampR pET vectors the β-lactamase gene is located downstream and in the same orientation as the T7 promoter. All pET translation vectors have the native T7 transcription terminator (Tφ) located before the β-lactamase gene. However, this terminator is only approximately 70% effective, allowing T7 RNA polymerase read-through to produce a small amount of β-lactamase RNA in addition to the target RNA. This results in the accumulation of β-lactamase enzyme in induced cultures. Accordingly, the orientation of the β- lactamase gene has been reversed in the pET-43.1 and pET-44 vectors, so that read-through by the T7 RNA polymerase will not result in increased levels of β-lactamase gene product.
I like to use the Promoter Hunter tool to help find promoters:
http://www.phisite.org/main/index.php?nav=tools&nav_sel=hunter
Software can only give predictions, but this tool seems to be fairly good at defining promoter sites.
Does this mean if someone ligates kanamycin resistance gene coding sequence (813 bp long) without its promotor?? into a plasmid vector (lets say TA cloning vector) and transform the bacteria with this kanamycin gene carrying plasmid, would the transformants be able to grow on kanamycin containing medium?
Douglas A Mitchell what drives the expression of kanamycin in pET-28a vector?
Subhash Verma Douglas A Mitchell That's exactly what's confusing me. " In kanR pET vectors, the kan gene is in the opposite orientation from the T7 promoter, so induction of the T7 promoter should not result in an increase in kan gene product. " Is it the T7 promoter which is driving the expression of KanR in pET28a or is it entirely different?
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