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pLKO.1 cloning (Kpn1 site)

pLKO.1 shRNA cloning

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#1 miST32

miST32

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Posted 08 January 2014 - 05:29 PM

Hey all,

I'm looking to create a lentiviral shRNA system for in-vivo transduction and understand the basics of cloning shRNA oligos into the pLKO.1 vector backbone.  (This is between the AgeI and EcoRI sites - for reference, here's the map:  http://www.addgene.org/8453/ )

 

In addition to the shRNA sequence, I'm looking to clone tissue-specific promoter and fluorophore 3' of the Puromycin resistance gene to generate my final vector.  There's a KpnI site there that I'd like to use, but I'm not versed enough in this vector to know if I'll be throwing something else out of frame - and there are a lot of important viral components in the vector that I'd rather not screw around with.

As far as I can tell, I wouldn't be inserting anything into any annotated ORFs, and I'm still within the TLRs for integration purposes.  So this seems okay at first glance.  Then again I tend to overlook things - so does anybody see an obvious problem with this idea?  

Thanks in advance - cheers!

 







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