Plasmid

Part:BBa_K5005005:Design

Designed by: YunQi Chen   Group: iGEM23_GEC-CHINA   (2023-10-05)


Primordial plasmid before point mutation.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 5150
    Illegal XbaI site found at 5659
    Illegal SpeI site found at 4780
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 5156
    Illegal SpeI site found at 4780
    Illegal NotI site found at 6646
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3111
    Illegal BglII site found at 6045
    Illegal BglII site found at 6342
    Illegal BglII site found at 8312
    Illegal BglII site found at 8378
    Illegal BglII site found at 8419
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 5150
    Illegal XbaI site found at 5659
    Illegal SpeI site found at 4780
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 5150
    Illegal XbaI site found at 5659
    Illegal SpeI site found at 4780
    Illegal NgoMIV site found at 7164
    Illegal NgoMIV site found at 8197
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3093
    Illegal BsaI site found at 8401
    Illegal BsaI.rc site found at 1534
    Illegal BsaI.rc site found at 6509
    Illegal BsaI.rc site found at 7117
    Illegal SapI site found at 451
    Illegal SapI.rc site found at 3742


Design Notes

NAMPT was cloned using PCR. Homologous arms play a crucial role in the Gibson assembly for connecting NAMPT with the vector. After obtaining the original pTarget-NAMPT plasmid, NAMPT will undergo site-directed mutagenesis at specific points to validate the impact of different mutations on NAMPT enzyme activity. Additionally, in the pTarget-NAMPT plasmid, NAMPT will be positioned downstream of the T7 promoter for directed evolution experiments on NAMPT. Here, you can clearly know that NAMPT is located downstream of the T7 promoter and can be edited by the editor to introduce mutations, resulting in NAMPT variants.


Source

Constructed in this work.

References

Zhu, X., Liu, H., Chen, L., Wu, C., Liu, X., Cang, Y., Jiang, B., Yang, X., & Fan, G. (2022). Addressing the Enzyme-independent tumor-promoting function of NAMPT via PROTAC-mediated degradation. Cell chemical biology, 29(11), 1616–1629.e12. https://doi.org/10.1016/j.chembiol.2022.10.007