Part:BBa_K747099:Design
Eukaryotic TAL expression plasmid
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 966
Illegal suffix found in sequence at 3901 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 966
Illegal SpeI site found at 3902
Illegal PstI site found at 3916
Illegal NotI site found at 972
Illegal NotI site found at 2146
Illegal NotI site found at 3909 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 966
Illegal BglII site found at 1563
Illegal BglII site found at 2914
Illegal BamHI site found at 2217
Illegal BamHI site found at 2920 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 966
Illegal suffix found in sequence at 3902 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 966
Illegal XbaI site found at 981
Illegal SpeI site found at 3902
Illegal PstI site found at 3916 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
further changes:
We removed all EcoRI, XbaI, SpeI, PstI, BsmBI, BbsI and PmeI restriction sites.
We replaced the BsaI restriction sites for inserting direpeats by BsmBI sites, because – according to the manufacturer - BsmBI is better suited for digest over one hour.
We added a consensus RBS in front of the ORF for expression in bacteria
We added a His-Tag to the n-terminal end to allow protein purification.
We flanked the whole sequence with the iGEM prefix and suffix.
Source
See MammoBrick for further information
The TAL open reading frame was synthesized by IDT.
The Plug and Play Effector Cassette was synthesized by IDT.
References
1. Scholze, H. & Boch, J. TAL effectors are remote controls for gene activation. Current Opinion in Microbiology 14, 47–53 (2011).
2. Moscou, M. J. & Bogdanove, A. J. A Simple Cipher Governs DNA Recognition by TAL Effectors. Science 326, 1501–1501 (2009).
3. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39, e82 (2011).
4. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nature Biotechnology 30, 460–465 (2012).
5. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nature biotechnology 29, 149–153 (2011).
6. Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nature Biotechnology 29, 143–148 (2010).
7. Boch, J. et al. Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors. Science 326, 1509–1512 (2009).
8. Liu, J. et al. Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy. Journal of Genetics and Genomics 39, 209–215 (2012).
9. Wood, A. J. et al. Targeted Genome Editing Across Species Using ZFNs and TALENs. Science 333, 307–307 (2011).
10. Sander, J. D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29, 697–698 (2011).
11. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nature Biotechnology 29, 695–696 (2011).
12. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nature Biotechnology 29, 731–734 (2011) 13. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012).