Part:BBa_K5196002:Design
THFMO Geneblock Fragment 1
Contents
Design Notes
We considered maintaining the integrity of the araBAD promoter site and gentamicin resistance sequence during our construct formation. We decided to use the EcoRI-HF and XbaI restriction sites to insert the THFMO sequence. However, we realized that by using the EcoRI-HF enzyme, we would be excising the consensus RBS (ribosome-binding site) AGGAGG, meaning the Gibson assembly must regenerate the Shine-Dalgarno sequence via the first THFMO fragment and its forward primer [2,3].
This fragment has a single-stranded overhang at its 3' end to allow for Gibson assembly with the 5' overhang of the THFMO fragment 2.
Source
The source of the THFMO gene is Pseudonocardia dioxanivorans strain CB1190 [1]. The pJN105-NicA2 plasmid was derived from Bordetella bronchiseptica; specifically, it is a derivative of the pBBR1MCS-5 plasmid originating from this organism.
References
[1] Sales, C. M., Grostern, A., Parales, J. V., Parales, R. E., & Alvarez-Cohen, L. (2013). Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species. Applied and Environmental Microbiology, 79(24), 7702-7708. https://doi.org/10.1128/AEM.02418-13
[2] Biolabs, N. E. (n.d.). Nebuilder. NEBuilder. https://nebuilder.neb.com/#!/add/
[3] Integrated DNA Technologies. IDT. (2024, September 11). https://www.idtdna.com/page
Sequence and Features:
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 384
Illegal PstI site found at 756 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 384
Illegal PstI site found at 756 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 161
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 384
Illegal PstI site found at 756 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 384
Illegal PstI site found at 756
Illegal AgeI site found at 564
Illegal AgeI site found at 907 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 609