Two, perhaps as you probably are aware, it is recommended that a stop codon be placed at the end of the insert. But as it turns out, most commercial plasmids come with stop codons at the end of the HIS tag and myc tag. Can't i simply forego my own stop codon and simply rely on those stop codons inserted by the company?
Hello,
You can do that if you want to actually use the tag (do not add a stop codon to the insert and this translation will proceed through the tag). If you do not want to use the tag add your own stop codon (more perferrably three in a row if you are expressing in e.coli in order to increase the termination efficiency) since then translation will not proceed through the tag.
Best wishes,
Hanna
The start codon is the first codon of a messenger RNA (mRNA) transcript translated by a ribosome. The start codon always codes methionine in eukaryotes and a modified Met (fMet) in prokaryotes. The most common start codon is AUG. However, naturally occurring non - AUG start codons have been reported for some cellular mRNAs. Bioinformatics programs usually allow for alternative start codons when searching for protein coding genes.
Hanna Alalam : (more perferrably three in a row if you are expressing in e.coli in order to increase the termination efficiency)
Do you often use multiple stop codons in your expression vectors? In that case are you using combination of the three?
Sylvain Julien I use on TAA since it is less prone to read-through than the other 2 also some strains have glnV44 which is a suppressor of UAG (although most expression strains do not have it) therefore it is preferable to use TAA.
P.S TAA is recognized by two release factors (RF1 and RF2) rather then one RF like the other two stop codons.
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