"conservative" mutagenesis withinin SV40 promotor

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Tue Feb 29 12:30:33 EST 2000


On 29 Feb 2000 13:32:52 -0000,
    Wolfgang Schechinger (Wolfgang.Schechinger at med.uni-tuebingen.de) wrote:

>In my vector (pCDNA3) the NeoR gene is driven by the SV40 
>promotor. Near the end of this promotor there is a XmaI site that 
>interferes with my cloning strategy. 
>Actually I'd use the quick change protocol in order to mutate the 
>sequence to destroy the XmaI site. It's very likely 
>that I'll destroy the promotor this way. 

I don't know much about the source of the "SV40 early promoter" in pcDNA3, 
but the XmaI site is not within the early promoter of SV40 (the virus).  
I have several constructions with the SV40 early promoter (isolated as 
a HindIII to KpnI fragment from SV40) that work just fine.

In pcDNA3 the XmaI site lies immediately outside of the HindIII site.
So you should be able to mutate it without affecting promoter
activity.

Regards,
Ashok

-- 
Ashok Aiyar
Assistant Professor                            Email: a-aiyar at nwu.edu
Department of Microbiology-Immunology            Tel: (312)  503 2524
Northwestern University, Chicago, IL 60611       Fax: (312)  503 1339




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