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His-tag fusion protein in pET-28a(+)

His-tag fusion protein in pET-28a(+) - Molecular Cloning Forum

His-tag fusion protein in pET-28a(+) - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 01-10-2010, 02:20 PM
Pipette Filler
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Default His-tag fusion protein in pET-28a(+)



Hi.
I am student only getting familiar with gene cloning.

I want to insert a gene in this vector (pET-28a(+), in order to get a his-tag. My question is: do you have to worry about the stop codon of the insert itself, because the vector already has a stop codon after the His-tag.

start--Histag---(start - Coding sequence -stop)----Histag--stop.

So, there are two stop codons, how do i know that translation does not stop before the his-tag is completely incorporated?

Thanks,
fragnatic
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Old 01-11-2010, 08:13 PM
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Default Re: His-tag fusion protein in pET-28a(+)

Those are not the same His tag. There is an N terminal, and optional C terminal tag if you do not incorporate a stop codon.
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fusion , fusionprotein , his-tag , histag , pet28a , protein , stop codon , vector


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