Plasmid

Part:BBa_K3600007

Designed by: yasmine koubaa   Group: iGEM20_EPFL   (2020-10-27)
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prTRX2-yomScarlet-I-tADH1


This cassette was assembled from 6 part plasmids via Golden Gate assembly using the BsmbBI restriction enzyme. The two connectors (pYTK002 and pYTK072), as well as the terminator (pYTK053) and the Ampicilin resistance/backbone (pYTK095) part plasmids are part of the YTK toolkit developed by Lee et al.1 and can be ordered online. The promoter part plasmid (BBa_K3600001) and the reporter part plasmid (BBa_K3600010) were synthesized by us.

Usage and Biology

This cassette contains the TRX2 promoter upstream of a reporter gene (mScarlet-i). TRX2 is a stress response gene in s.cerevisiae that is regulated by the YAP1 master regulator2. This cassette is then assembled with a part plasmid containing Lys2 homology sequences (BBa_K36000012) in another Golden Gate BsmBI assembly, to give a final integration cassette (BBa_K3600014).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 936
    Illegal EcoRI site found at 1666
    Illegal XbaI site found at 945
    Illegal XbaI site found at 1843
    Illegal SpeI site found at 2811
    Illegal PstI site found at 2820
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 936
    Illegal EcoRI site found at 1666
    Illegal SpeI site found at 2811
    Illegal PstI site found at 2820
    Illegal NotI site found at 924
    Illegal NotI site found at 2830
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal suffix found in sequence at 2413
    Illegal EcoRI site found at 936
    Illegal EcoRI site found at 1666
    Illegal BglII site found at 2284
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 936
    Illegal EcoRI site found at 1666
    Illegal XbaI site found at 945
    Illegal XbaI site found at 1843
    Illegal SpeI site found at 2811
    Illegal PstI site found at 2820
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 936
    Illegal EcoRI site found at 1666
    Illegal XbaI site found at 945
    Illegal XbaI site found at 1843
    Illegal SpeI site found at 2811
    Illegal PstI site found at 2820
    Illegal NgoMIV site found at 2768
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4, 975–986 (2015).

[2] Kuge S, Jones N. YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. The EMBO journal. 1994 Feb;13(3):655-64.

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