Part:BBa_K5196015:Design

ALDH Gibson Primer 2

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 primer regenerates the 3’ overhang of ALDH Fragment 1.

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
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 19
  
• 1000
  COMPATIBLE WITH RFC[1000]