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SD sequence of di-cistronic cloning
Posted by: Rosalind324 (IP Hidden, New member, 9)
Date: May 12, 2006 07:40AM
I wonna construct a di-cistronic cloning, pET-22b(+) vector, and insert gene II behind gene I (both need SD sequence and termination codon),
but I was so confused. How can I determine the place (the bp length)between the termination codon of gene I and SD sequence at the begining of gene II?no place(follow on the heels of gene I) or add some inessential base pairs? Any suggestion?or some protocols recommended? Thank in advice.
Re: SD sequence of di-cistronic cloning
Posted by: Rosalind324 (IP Hidden, New member, 9)
Date: May 12, 2006 09:27AM
I have consult some series of non-fusion vectors such as pBAT4 (GST, non-fusion) which has two promoter with directon conversely
In fact, I just want to construct two-gene expression cloning with one promoter, because I hope gene I protein ahead could help gene II protein soluble, just in the manner of co-expression. and I have no idea how much interval between them.That 's the key of the question. by the way, all thing I mentioned above is about prokaryote(E.coli). Thank in advice.
Re: SD sequence of di-cistronic cloning
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: May 12, 2006 02:15PM
If co-expression gene 1 and 2 is your only requirement, you can try IRES bicistronic vectors such as pIRES from clontech. Just clone your gene of interest at MCS1 and the helper gene downstream of IRES sequence at MCS2.
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