Part:BBa_K5196012:Design

ALDH Geneblock Fragment 1

Design Notes

We considered maintaining the integrity of the T7 promoter site and kanamycin resistance sequence during our construct formation. We decided to use the XbaI and PspXI restriction sites to insert the ALDH sequence [2,3].

This fragment’s overhangs are generated by the ALDH Gibson Primers 1 and 2.

Source

The source of the ALDH gene is Pseudonocardia dioxanivorans strain CB1190 [1,4]. The pET-28-NicA2 plasmid was derived from E. coli.

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

[4] Database, A. P. S. (2022, November 1). Alphafold protein structure database. https://alphafold.ebi.ac.uk/entry/Q88CR0

Sequence and Features:

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 507
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 507
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 519
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 507
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 507
  Illegal NgoMIV site found at 341
  Illegal NgoMIV site found at 450
  Illegal NgoMIV site found at 777
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 231
  Illegal SapI.rc site found at 146