I use homologous recombination cloning to assemble CMV-cDNA-PolyA (3.9 bp) to AAV transfer vector. After sequencing, I found the fragment is reversely inserted between 5'ITR and 3'ITR.
We have hesitation about the next step to produce the rAAV via a triple plasmid HEK293 system.
I am wondering:
(1) whether this vector is producible via triple plasmids HEK293 system?
(2) titer is detectable via designing primer pairs on CMV?
(3) cDNA can be expressed in rAAV genome and detected by qPCR (SYBR Green)?