Difference between revisions of "Part:BBa K2446041:Design"
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===Source=== | ===Source=== | ||
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===References=== | ===References=== | ||
Revision as of 07:44, 31 October 2017
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===