Difference between revisions of "Part:BBa K2446041:Design"

 
 
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===Design Notes===
 
===Design Notes===
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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.
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<B>OE-PCR:</B> 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.
  
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1.Use proofreading enzyme for extension.
  
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2.Run 10-15 PCR cycles without end primers. (Template extension step)
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3.Add end primers, then continue cycling for another 15-20 rounds.
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4.Gel extract the correct fragment. Clone into a vector.
  
 
===Source===
 
===Source===
  
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The three fragments are from IDT.
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===References===
 
===References===
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[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).
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[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).
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[3] Chen, X., Zaro, J. L. & Shen, W.-C. Fusion protein linkers: property, design and functionality. Advanced drug delivery reviews 65, 1357-1369 (2013).
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[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).

Latest revision as of 07:49, 31 October 2017


ZF_43-8_KRAB


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 301
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 184
    Illegal AgeI site found at 19
  • 1000
    COMPATIBLE 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).