Part:BBa_K5196004:Design

THFMO Geneblock Fragment 3

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 5' end to allow for Gibson assembly with the 3' 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:

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 13
  Illegal BamHI site found at 608
  Illegal XhoI site found at 63
  Illegal XhoI site found at 1382
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 750
  Illegal NgoMIV site found at 1084
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 672
  Illegal BsaI.rc site found at 984