Part:BBa_K1329001
Hag-KpnI
The designed sequence was synthesized by IDT (Integrated DNA Technologies).
Application
As the original Flagellin gene (hag) contains multiple forbidden iGEM restriction sites the complete sequence of Hag-KpnI was synthesized by IDT. We inserted the KpnI restriction site because it does not occur in the iGEM prefix and suffixes of the shipping vector pSB1C3. This Brick was designed as a platform for the insertion of additional domains via Gibson assembly after linearizing the iGEM backbone pSB1C3-Hag-KpnI. The modified Flagellin can be implemented into Bacillus subtilis.
Examples
- Cup1-1
- D2-Strep
Test restriction with EcoRI and PstI:
Constructs were created with Gibson assembly. 5 clones were tested for the insertion of the fragment.
For the insertion of the Cup1 fragments three clones could be considered as positive. The 5 D2-Strep clones were presumably positive since the D2-domain contained an additional PstI restriction site that had to be removed later on.
An additional PCR was performed with the isolated plasmids and primers that bind in the inserted domain and Flagellin suffix. The PCR product further confirmed that the domains have been inserted.
In the end the constructs were sequenced which finally confirmed that Hag-KpnI can be used as a platform for the insertion of multiple different domains into the Flagellin of Bacillus subtilis.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 588
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