Plasmid_Backbone

Part:BBa_K5005002:Design

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


The BFP protein is located downstream of the T7 promoter.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 8002
    Illegal NheI site found at 315
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 1053
    Illegal NotI site found at 8008
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 8002
    Illegal BglII site found at 2719
    Illegal BglII site found at 2785
    Illegal BglII site found at 2826
    Illegal BglII site found at 6250
    Illegal BamHI site found at 1047
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 8002
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 8002
    Plasmid lacks a suffix.
    Illegal XbaI site found at 8017
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 1571
    Illegal NgoMIV site found at 2604
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 2808
    Illegal BsaI site found at 6232
    Illegal BsaI.rc site found at 1524
    Illegal BsaI.rc site found at 4673
    Illegal SapI site found at 3590
    Illegal SapI.rc site found at 6881


Design Notes

The Target plasmid expresses the target gene BFP (blue fluorescence), derived from site-directed mutagenesis of EGFP, which has the potential to be evolved back into EGFP by the Editor. BFP is positioned downstream of the T7 promoter, enabling specific editing of EBFP by the Editor. If the TRACE system operates effectively, it is feasible to observe cells where blue fluorescence is converted into green fluorescence.

Primer EGFP-F:atagggattccagagctagcgaatttatggtgagcaagggcgagga and EGFP-R: tgtagtctgcggccgcggatccttacttgtacagctcgtccatgcc were used to insert EGFP(BBa_K5005001) into pTarget (BBa_K5005012). Primer BFP-F:acttcaagatccgccacaacatcgaggacggcagcgtgc and BFP-R:tgtggcggatcttgaagttcaccttgatgccgttcttctgct were used to converted EGFP(BBa_K5005001) to BFP (BBa_K5005000).

Source

Constructed in this work.

References

Chen, H., Liu, S., Padula, S., Lesman, D., Griswold, K., Lin, A., Zhao, T., Marshall, J. L., & Chen, F. (2020). Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. Nature biotechnology, 38(2), 165–168. https://doi.org/10.1038/s41587-019-0331-8