how many stop codons do I need?

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how many stop codons do I need?

Postby vibriotko » Mar 08 2011 3:33 pm

Hi all. 300bps into an ampicillin cassette sequence, I want to insert a stop codon followed by a start codon to express the full chloramphenicol cassette sequence. how many stop codons do I need to code between the ampicillin peptide and the n-terminal of the full Chloramphenicol cassette to make sure the Chloramphenicol cassette expresses?
Thanks.
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Re: how many stop codons do I need?

Postby relaxin » Mar 08 2011 5:02 pm

If the STOP codon is in-frame with the ampicillin resistant gene coding sequence, one STOP codon should be able to stop the translation. To translate a bicistronic coding sequence (in your case, Chloramphenicol resistant gene), you will need a RBS in front of the ATG codon of the chloramphenicol resistant gene.
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Re: how many stop codons do I need?

Postby vibriotko » Mar 09 2011 8:54 am

Do I need spacer DNA between the stop codon and the RBS sequence?
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Re: how many stop codons do I need?

Postby relaxin » Mar 09 2011 9:15 am

Yes, but the length required is not clear-cut. However, the spacer between the RBS and the ATG of the second gene has to be short (7-11 bp).
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Re: how many stop codons do I need?

Postby vibriotko » Mar 09 2011 10:30 am

I am seeing a shine-delgado sequence in prokaryotes of AGGAGGT. But in novagens pET vectors, I see a sequence of AAGGAG. What rbs sequence should I use?
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Re: how many stop codons do I need?

Postby relaxin » Mar 09 2011 12:16 pm

I do not think it matters. I noticed some commercial vectors use AAGGAG, AAGGAA, or GAGGAA.
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Re: how many stop codons do I need?

Postby Lullo » Mar 16 2011 5:35 am

42
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Re: how many stop codons do I need?

Postby mdfenko » Mar 16 2011 11:25 am

Lullo wrote:42

that's the answer to (life, the universe and) everything
talent does what it can
genius does what it must
i do what i get paid to do
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