Coding

Part:BBa_K5195002:Design

Designed by: Vanessa Chiprez Meza   Group: iGEM24_Stanford   (2024-09-29)


DUX4-DBD (DNA Binding Domain)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 35
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 35
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 191
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 35
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 35
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 97


Design Notes

Design: The DUX4-DBD sequence is an isoform of DUX4-FL that contains 217 amino acids, slightly longer than the 157 amino acid sequence known as DUX4-S. [3]

Characterization: Part characterization for the KRAB domain was performed using the composite part BB_Ka (DUX4-DBD linked to a mNeonGreen fluorophore).

Mitsuhashi, H., Ishimaru, S., Homma, S., Yu, B., Honma, Y., Beermann, M. L., & Miller, J. B. (2018). Functional domains of the FSHD-associated DUX4 protein. Biology Open, 7(The Company of Biologist Ltd). https://doi.org/10.1242/bio.033977


Source

Through our initial tet-inducible DUX4-FL plasmid, AddGene pCW57.1-DUX4-CA, we yielded our DBD sequence. In cloning the construct, the backbone excluded the transcription-activating domain, so a DUX4 gene with 217 amino acids.

Sequence sourced from the following references: [1] Model systems of DUX4 expression recapitulate the transcriptional profile of FSHD cells. Jagannathan S, Shadle SC, Resnick R, Snider L, Tawil RN, van der Maarel SM, Bradley RK, Tapscott SJ. Hum Mol Genet. 2016 Oct 15;25(20):4419-4431. doi: 10.1093/hmg/ddw271. 10.1093/hmg/ddw271 PubMed 28171552 [2] U.S. National Library of Medicine. (n.d.). Dux4 double homeobox 4 [homo sapiens (human)] - gene - NCBI. National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/gene/100288687


References