Reading Frame Shift After Cloning

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July 20, 2021
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July 20, 2021


E. coli expression, reading frame shift after cloning into pET28 vector?

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The easiest solution would be adding two more bases after the G to get a second codon after the ATG (e.g. addition of “GN” would give you a glycine after the Met which hopefully is small enough not to disturb your proteins function). Adding an additional start codon which are not even in frame like you suggested will give you a mixture of two different proteins (one probably shorter one starting at the first ATG and stopping somewhere at the first stop codon in its frame in the sequence, and your target protein)

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