Primer

Part:BBa_K5196005:Design

Designed by: Avi Patel   Group: iGEM24_Michigan   (2024-09-30)


THFMO Gibson Primer 1

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 regenerates the 5' overhang of the THFMO fragment 1 as well as the Shine-Dalgarno sequence, allowing for the recombination of the pJN105-NicA2 plasmid into the pJN05-THFMO plasmid.

The annealing temperature of this primer is 69.3°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:


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 14
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]