Project

Part:BBa_K2865002:Design

Designed by: Chuqi Wang   Group: iGEM18_SMMU-China   (2018-09-29)
Revision as of 15:09, 30 September 2018 by William Wang (Talk | contribs)


[AAV9]-Left-ITR


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

ITR regions are GC rich and have extensive secondary structures. The sequences of left and right ITRs are symmetry, 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 factors above, we decided to employ nested PCR 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. Point mutation was performed to remove Pst1 illegal site. The final product was a little bit longer than 145 bases, because the primers initiated from the outside of ITR region.


Source

This part was cloned from an AAV-9 shuttle vector.

References