Difference between revisions of "Part:BBa K1638004:Design"
Jpettersen (Talk | contribs) (→Source) |
Jpettersen (Talk | contribs) (→Design Notes) |
||
| Line 19: | Line 19: | ||
2) Amplify through PCR with designed primers | 2) Amplify through PCR with designed primers | ||
| − | 3) Digest PCR-product and pSB1C3- | + | 3) Digest PCR-product and pSB1C3-T18 with BamHI and PstI. |
4) Ligate the two digested product. | 4) Ligate the two digested product. | ||
Revision as of 15:35, 20 July 2015
T18 domain of CyaA from Bordetella pertussis
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 550
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 91
Illegal NgoMIV site found at 501
Illegal AgeI site found at 307 - 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