Difference between revisions of "Part:BBa K3505024"

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<partinfo>BBa_K3505024 short</partinfo>
 
<partinfo>BBa_K3505024 short</partinfo>
  
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===Usage And Biology===
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B-arrestin has the ability to normally bind to the GPCR receptor and facilitate its endocytosis, desensitization or GPCR-independent signaling effects. The second composite of our TANGO system is a fusion of the b-arrestin-2 with the TEV protease.TEV protease recognizes and cleaves the cleavage site. This low proximity allows the TEV protease that is tagged to the βarrestin-2 carrier to cleave its substrate (TCS) .
  
  
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===Verification of Cloning===
 
===Verification of Cloning===
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[[File:T--Thessaly--b-arrestin-digestion.png|700px|thumb|none|<i><b>Fig.1:</b>(U=Uncut C=Cut) Restriction Digestion of b-arrestin-TEV protease with SlaI, Expected bands : 2014 bp + 892 bp + 760 bp + 394 bp, Positive Result: green arrows</i>]]
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[[File:T--Thessaly--b-arrestin-digestion.png|700px|thumb|none|<i><b>Fig.3:</b>(U=Uncut C=Cut) Restriction Digestion of b-arrestin-TEV protease with SlaI, Expected bands : 2014 bp + 892 bp + 760 bp + 394 bp, Positive Result: green arrows</i>]]
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===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K3505024 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3505024 SequenceAndFeatures</partinfo>

Latest revision as of 00:36, 28 October 2020


B-arrestin:TEV Protease GB compatible with B2-B5

Usage And Biology

B-arrestin has the ability to normally bind to the GPCR receptor and facilitate its endocytosis, desensitization or GPCR-independent signaling effects. The second composite of our TANGO system is a fusion of the b-arrestin-2 with the TEV protease.TEV protease recognizes and cleaves the cleavage site. This low proximity allows the TEV protease that is tagged to the βarrestin-2 carrier to cleave its substrate (TCS) .


Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is presents in pUPD2 and has overhangs compatible for Golden Braid cloning. The CDS has position B2-B5.

Figure 1.The overhangs of this part in the GoldenBraid Grammar.


Figure 2. The level 0 module : pupd2-b-arrestin-TEV protease (illustration from SnapGene)

Verification of Cloning

Fig.3:(U=Uncut C=Cut) Restriction Digestion of b-arrestin-TEV protease with SlaI, Expected bands : 2014 bp + 892 bp + 760 bp + 394 bp, Positive Result: green arrows

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 1611
    Illegal PstI site found at 303
    Illegal PstI site found at 390
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 1611
    Illegal PstI site found at 303
    Illegal PstI site found at 390
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 203
    Illegal XhoI site found at 600
    Illegal XhoI site found at 994
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 1611
    Illegal PstI site found at 303
    Illegal PstI site found at 390
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 1611
    Illegal PstI site found at 303
    Illegal PstI site found at 390
    Illegal AgeI site found at 586
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1545
    Illegal SapI.rc site found at 1893