Coding

Part:BBa_K2374003

Designed by: Qian Zeng   Group: iGEM17_Tongji_China   (2017-10-20)
Revision as of 19:34, 31 October 2017 by Amang Z (Talk | contribs) (→‎Design Notes)


ple (Tyrosine 3-monooxygenase, TH) -> (fruit fly)

TH
Use in D.melanogaster
RFC standard RFC 10 compatible
Backbone pSB1C3
Submitted by [http://2017.igem.org/Team:Tongji_China Tongji_China 2017]

ple (Tyrosine 3-monooxygenase, TH)

ple (TH) is a rate-limiting enzyme in the dopamine’s synthesis, and it plays an important role in the physiology of adrenergic neurons.
Catalytic activity: L-tyrosine + tetrahydrobiopterin + O2 = L-dopa + 4a-hydroxytetrahydrobiopterin.
This subpathway is part of the pathway dopamine biosynthesis, which is itself part of catecholamine biosynthesis.


dopa


Design Notes

ple has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.

标题

According to our experiment results to judge, the ple coding sequence is hard to clone from Drosophila 's cDNA library because of its multi-segment repeats. So we recommend that you obtain from the constructed plasmid, or synthesize it directly.

We ordered a synthetic ple from GENEWIZ®, and cloned it into pUAST vector with two restriction sites: EcoRI and XbaI.

We cloned UAS-TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-UAS-TH.

标题

We also did two site direct mutagenesis for submission:
EcoR I (130) GAATTC->AAATTC
Pst I (782) CTGCAG->CTGCTG

Source

GeneCards®: The Human Gene Database [http://www.genecards.org/cgi-bin/carddisp.pl?gene=TH]

NCBI [1]

FlyBase [http://flybase.org/reports/FBgn0005626.html]

References

Janice A. Fischer, et al. GAL4 activates transcription in Drosophila. Nature 332, 853 - 856 (1988)

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 13
    Illegal PstI site found at 256
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 13
    Illegal PstI site found at 256
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1540
    Illegal XhoI site found at 672
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 13
    Illegal PstI site found at 256
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 13
    Illegal PstI site found at 256
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 918
    Illegal BsaI site found at 1884
    Illegal BsaI.rc site found at 636
    Illegal BsaI.rc site found at 1402
    Illegal BsaI.rc site found at 1551
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