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!