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===Functional Parameters===
 
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Latest revision as of 02:58, 28 October 2020


prGLR1-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_K3600004) and the reporter part plasmid (BBa_K3600010) were synthesized by us.

Usage and Biology

This cassette contains the GLR1 promoter upstream of a reporter gene (mScarlet-i). GLR1 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_K3600017).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 935
    Illegal EcoRI site found at 1665
    Illegal XbaI site found at 944
    Illegal XbaI site found at 1272
    Illegal XbaI site found at 1842
    Illegal SpeI site found at 2810
    Illegal PstI site found at 2819
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 935
    Illegal EcoRI site found at 1665
    Illegal SpeI site found at 2810
    Illegal PstI site found at 2819
    Illegal NotI site found at 923
    Illegal NotI site found at 2829
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal suffix found in sequence at 2412
    Illegal EcoRI site found at 935
    Illegal EcoRI site found at 1665
    Illegal BglII site found at 2283
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 935
    Illegal EcoRI site found at 1665
    Illegal XbaI site found at 944
    Illegal XbaI site found at 1272
    Illegal XbaI site found at 1842
    Illegal SpeI site found at 2810
    Illegal PstI site found at 2819
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 935
    Illegal EcoRI site found at 1665
    Illegal XbaI site found at 944
    Illegal XbaI site found at 1272
    Illegal XbaI site found at 1842
    Illegal SpeI site found at 2810
    Illegal PstI site found at 2819
    Illegal NgoMIV site found at 2767
  • 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] Grant CM, MacIver FH, Dawes IW. Stationary‐phase induction of GLR1 expression is mediated by the yAP‐1 transcriptional regulatory protein in the yeast Saccharomyces cerevisiae. Molecular microbiology. 1996 Nov;22(4):739-46.