Part:BBa_K2865003:Design
[AAV9]-Right-ITR
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 34
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
ITR regions are GC rich and have extensive secondary structures. The sequences of left and right ITRs are identical but inverse with several palindromic sequences inside, making the designed PCR primers easy to bind to the incorrect sites. Besides the complex structures, there's also a Pst1 restriction site in both left and right ITR regions which is illegal and required to be deleted. Considering all the negative factors mentioned above, we decided to employ nested PCR along with single point mutation to amplify ITRs. To tackle the problem of non-specific binding, two pairs of primers were desigend: the first was far from ITR core region and the second was approximal. In the inner pair of primers, one nucleotide was changed so that Pst1 restriction site could be removed. The final product was a little bit longer than 145bp, because the primers initiated from the outside part of ITR region, but this extension does not influence the functions of ITRs.
Source
This part was cloned from an AAV-9 shuttle vector.