I want to clone a human gene in pET-21a and transfer to E.coli. I cloned the gene in the Nde1 site and it’s sequence is CA/TATG My question is, does the ATG of Nde1 act as a start codon and as a result must I delete the ATG of my sequence or I can use the ATG of the vector in the Nde1 site as a start codon? (NOTE: My gene is eukaryotic).
Yes you have to use the ATG of NdeI as the start codon and design the the primer in a may to contain only one start codon after ligation. two ATG are not required for any genes (eukaryote/prokaryote).
Dear Ananda thanks for your answer I have any question, when we clone eucaryotic gene in E.coli is it necessary we use kozak site before .my opinion is that, Ecoli has Shine-Dalgarno sequence ,and pET also has this sequence in result it's not necessary ,how about your opinion?
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Hi,
Keep it simple, the expression system (host) is E.coli, so only E.coli RNA polymerase is going to trigger the transcription of recombinant DNA cloned in any pET vectors. Just forget about that this a eukaryotic gene at this point and clone the coding sequence in between the restriction sites of the vector and make sure the reading frames are correctly manipulated.
You may want to read about pET system, I am attaching herewith.
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