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Question: Can I cut out rb glob PA Terminator

cloning dna plasmid restriction enzyme

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#1 Fabian Yu

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Posted 21 January 2015 - 09:11 AM

Hi There,

 

I am doing a cloning project with the pCAGGs promoter.  I already ligated a fragment of my gene of interest  in the plasmid, and now I need to add an IRES::Puro sequence.  So far my ligation is not working but I'm not sure whether it is b/c I cut out the rb glob PA Terminator in the plasmid.  I know the poly a site helps with transcription but is it essential for my plasmid? Only reason I did that was due to the cut site  issue 

 

thank

 

 



#2 CPRES

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Posted 21 January 2015 - 06:23 PM

Looks like you are making a bicistronic vector: gene of your interest (with stop codon but without polyA signal)-IRES-puro-polyA.

 

If so, you do need polyA at the end of IRES-puro cassette.


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