Plasmid

Part:BBa_K5196011:Design

Designed by: Malik Alabbas   Group: iGEM24_Michigan   (2024-10-01)


ALDH Expression Plasmid

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].

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 EcoRI site found at 333
    Illegal PstI site found at 7558
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 333
    Illegal NheI site found at 116
    Illegal PstI site found at 7558
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 333
    Illegal BglII site found at 222
    Illegal BglII site found at 6981
    Illegal BamHI site found at 413
    Illegal BamHI site found at 714
    Illegal BamHI site found at 1663
    Illegal XhoI site found at 1416
    Illegal XhoI site found at 1859
    Illegal XhoI site found at 7570
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 333
    Illegal PstI site found at 7558
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 333
    Illegal PstI site found at 7558
    Illegal NgoMIV site found at 453
    Illegal NgoMIV site found at 2158
    Illegal NgoMIV site found at 5201
    Illegal NgoMIV site found at 5361
    Illegal NgoMIV site found at 6949
    Illegal NgoMIV site found at 7392
    Illegal NgoMIV site found at 7501
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 7282
    Illegal SapI.rc site found at 4283
    Illegal SapI.rc site found at 7197