Coding
ThTdT

Part:BBa_K5228000:Design

Designed by: Narjis Alhusseini   Group: iGEM24_UBC-Vancouver   (2024-09-01)


Thermostable Terminal Deoxynucleotidyl Transferase (ThTdT)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 275
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 275
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 275
    Illegal BglII site found at 164
    Illegal BglII site found at 431
    Illegal BglII site found at 799
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 275
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 275
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The following 30 base-pair overhang primers were used for Gibson assembly of the ThTdT part into the pET-28b plasmid backbone.

Forward (P3, Fig. 2.2): 5'-TTCCTTTCGGGCTTTGTTAGCAGCCGGATCCTAAGCATTTCTTTCCCATGGTTCA-'3

Reverse (P4, Fig. 2.2): 5'-ATTTTGTTTAACTTTAAGAAGGAGATATACATGGGCAGCAGCCATCATC-3'


Source

The gene sequence was derived from an E. coli TdT mutant library generated by Chua et al.(2020).

References

Chua, J. P. S., Go, M. K., Osothprarop, T., Mcdonald, S., Karabadzhak, A. G., Yew, W. S., Peisajovich, S., & Nirantar, S. (2020). Evolving a Thermostable Terminal Deoxynucleotidyl Transferase. ACS synthetic biology, 9(7), 1725–1735. https://doi.org/10.1021/acssynbio.0c00078