Difference between revisions of "Part:BBa K2374003:Design"

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===Design Notes===
 
===Design Notes===
  
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'''ple''' has three variable splice mRNA products. We choose isoform B to construct our plasmid.  
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'''ple''' has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.  
  
 
[[File:2017tongji_image_registry_TH_base.png|center|300px|标题]]
 
[[File:2017tongji_image_registry_TH_base.png|center|300px|标题]]
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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.  
 
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.
 
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 ordered a synthetic '''ple''' from GENEWIZ®, and cloned it into pUAST vector with two restriction sites: EcoRI and XbaI.
  
 +
We cloned TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-TH.
  
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We cloned UAS-TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-UAS-TH.
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[[File:2017tongji_registry_image_UTH.png|center|300px|标题]]
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+
−
[[File:2017tongji image registry UASTHtest.png|center|400px|标题]]
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−
 
+
  
 
We also did one site direct mutagenesis for submission:<br>
 
We also did one site direct mutagenesis for submission:<br>
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Pst I (647) CTGCAG->CTGCTG
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EcoR I (130) GAATTC->AAATTC
 +
Pst I (782) CTGCAG->CTGCTG
  
 
===Source===
 
===Source===

Latest revision as of 03:05, 2 November 2017


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1157
    Illegal XhoI site found at 289
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 535
    Illegal BsaI site found at 1501
    Illegal BsaI.rc site found at 253
    Illegal BsaI.rc site found at 1019
    Illegal BsaI.rc site found at 1168


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

标题

We also did one 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)