I'm trying to design a DNA/plasmid construct for eventual protein expression in E. coli. I wanted to know if there is restriction enzyme with a recognition site that, when cut, gives a stop codon, preferably TAA or TGA - either by blunt cutting or filling in of a 5' overhang ( of course it needs also to be in-frame). I have been searching for a while now and cant seem to come up with anything. Has anyone had experience with any such enzyme or is anyone familiar with software that can search for such an enzyme.
It's kind of a tricky question... For an enzyme to generate a stop codon after cut, it is because the stop codon is already part of the recognition site. So, if you are looking for enzymes which recognition sequences contain stop codons (PacI is one of them), you can check the Promega site: http://www.promega.com/resources/product-guides-and-selectors/restriction-enzyme-resource/restriction-enzyme-reference-information/
which has a section titled: "Restriction Enzyme Recognition Sequences Containing Start and Stop Codons"
If designing a vector for cloning and expression of coding sequences, wouldn't it be easier to include stop codons in all three possible reading frames downstream of the cloning site in the vector, so regardless of the enzyme you are using for cloning, you make sure your protein will end in any of those stop codons? Let´s say: tgaATCGtagATACtga
That is how it is usually done for expression vectors.
That's a tall order. If you fill-in, you'll get the same sequence when you perform blunt end-ligation. Try searching the NEB restriction enzymes for TAA or TGA and select the radio button to include all ambiguities. Something that might work is using AgsI and using 3' exonuclease to trim down, then blunt end getting TAA. You're going to be constrained by the sequence requirements of the enzymes. Good luck.
Thanks Jason - I found that PacI gives me a TAA site when cut, treated with Klenow of T4 polymerase and re-ligated. The only thing is that everything downstream of TAA is out of frame but this is fine for my purposes
It has a TAA site before as well. It should work as long as the frame shift pops the second TAA into the correct frame to make the stop.
Good luck.
Unfortunately I was solving the other problem (removing STOP codon), so I cannot tell you any specific RE for this.
If you introduce STOP codon, why do you bother about being in-frame?
It's kind of a tricky question... For an enzyme to generate a stop codon after cut, it is because the stop codon is already part of the recognition site. So, if you are looking for enzymes which recognition sequences contain stop codons (PacI is one of them), you can check the Promega site: http://www.promega.com/resources/product-guides-and-selectors/restriction-enzyme-resource/restriction-enzyme-reference-information/
which has a section titled: "Restriction Enzyme Recognition Sequences Containing Start and Stop Codons"
If designing a vector for cloning and expression of coding sequences, wouldn't it be easier to include stop codons in all three possible reading frames downstream of the cloning site in the vector, so regardless of the enzyme you are using for cloning, you make sure your protein will end in any of those stop codons? Let´s say: tgaATCGtagATACtga
That is how it is usually done for expression vectors.
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