Part:BBa_K2446041:Design
ZF_43-8_KRAB
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 301
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 184
Illegal AgeI site found at 19 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
This part was a fusion protein which assembled by three fragments OE-PCR. It contain three core domains from N-terminal to C-terminal: ZF_43-8 DNA binding domain (DBD), nuclear location sequence and transcription regulating domain.
OE-PCR: PCR amplify and connecting the necessary fragments, using proofreading polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temperature. Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.
1.Use proofreading enzyme for extension.
2.Run 10-15 PCR cycles without end primers. (Template extension step)
3.Add end primers, then continue cycling for another 15-20 rounds.
4.Gel extract the correct fragment. Clone into a vector.
Source
The three fragments are from IDT.
References
[1] Lohmueller, J. J., Armel, T. Z. & Silver, P. A. A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Nucleic Acids Research 40, 5180--5187 (2012).
[2] Witzgall, R., O'Leary, E., Leaf, A., Onaldi, D. & Bonventre, J. V. The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proceedings of the National Academy of Sciences 91, 4514-4518 (1994).
[3] Chen, X., Zaro, J. L. & Shen, W.-C. Fusion protein linkers: property, design and functionality. Advanced drug delivery reviews 65, 1357-1369 (2013).
[4] A. Klug, The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annual review of biochemistry 79, 213--231 (2010).