Hi, I am trying to synthesise a custom DNA construct.
My end of DNA construct has a 6x His-tag attached for purification later on. However, I am not sure if I should include the stop codon before or after the 6x His-tag.
Also, I will be cloning the sythesised construct into a different plasmid and I will need to add restriction sites. Do I put the restriction sites after the stop codon or before?
Thank you in advanced.
Adam B Shapiro
If you want the C-terminal His-tag to be part of the expressed protein, the stop codon should come at the 3' end of the gene, i.e., after the His tag.
The restriction sites should be outside the open reading frame, which includes the stop codon.
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