Part:BBa_K5196008:Design
THFMO Gibson Primer 4
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 primer does not regenerate overhangs for the THFMO Fragment 2.
The annealing temperature of this primer is 64.4°C.
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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 13
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]