Difference between revisions of "Part:BBa K1638032:Design"
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===Source=== | ===Source=== | ||
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===References=== | ===References=== | ||
| + | [1]: Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(10):5752-6. | ||
Latest revision as of 00:38, 18 September 2015
T18 domain of cyaA with cAMP-induced RFP generator
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1719
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1260
Illegal NgoMIV site found at 1670
Illegal AgeI site found at 712
Illegal AgeI site found at 824
Illegal AgeI site found at 1476 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Addition of a protein coding domain to the suffix by Standard Assembly RFC[10] creates a scar-site. As the scarsite encodes a stop codon (uacuag), this will leave the fused protein coding domain untranslated.
Instead we recommend the following assembly method:
1) Design primers for amplification of the C-terminal fusion protein with BamHI resitriction site included in the forward primer. The primers must also include prefix and suffix.
1.2) Forward primer: 5'-ATATGGATCCNNN...NNN-3'
1.3) Reverse primer: 5'-ATATCTGCAGCGGCCGCTACTAGTANNN..NNN-3'
2) Amplify through PCR with designed primers
3) Digest PCR-product and pSB1C3-T18 with BamHI and PstI.
4) Ligate the two digested product.
Source
pUT18C
References
[1]: Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(10):5752-6.