GFP fusion mamm. cells

Joris Heus jheus at ruly46.medfac.leidenuniv.nl
Fri Jan 24 03:04:06 EST 1997


Hello,

Maybe someone could help me with the following gene expression problem:

The aim is to make a ES-knockout construct to target the first exon of the
Duchenne Muscular Dystrophy gene and simultaneously introduce GFP. 
I would like to make a fusion construct between the DMD 5' untranslated
region (exon 1) right up to the ATG and GFP right from the ATG. The plan
is to use ApaI (only site available) to join both fragments.

The question is where to put this site with respect to the ATG, without
inhibiting the normal DMD promoter/expression by disturbing important
sequences like the Kozak consensus. I will be joining DMD and GFP
sequences as PCR fragments, so I am relatively free in the design of
primers and thus where to put the ApaI site.  

Welcoming all suggestions,

					Joris Heus


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"  Joris Heus					"
"  Leiden University				"
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