Difference between revisions of "Part:BBa K3505033"

(→‎Design Notes)
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The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in alpha1R vector <bbpart>BBa_K3505008</bbpart> and has overhangs compatible for GoldenBraid cloning.
 
The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in alpha1R vector <bbpart>BBa_K3505008</bbpart> and has overhangs compatible for GoldenBraid cloning.
  
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[[Image:T--Thessaly--ANDERSON-TETR-PHOT.png|900px|thumb|none|<I><b>Figure 2.</b> The level A module of Tet-Off system : AndersonJ23115:RBS-TetR-Double terminator </i>]]
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[[Image:T--Thessaly--ANDERSON-TETR-PHOT.png|900px|thumb|none|<I><b>Figure 1.</b> The level A module of Tet-Off system : AndersonJ23115:RBS-TetR-Double terminator </i>]]
  
 
===Verification of Cloning===
 
===Verification of Cloning===
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[[File:T--Thessaly--Anderson--TETR-LEVEL1.png|700px|thumb|none|<i><b>Fig.1:</b>(U=Uncut C=Cut) Restriction digestion of T: AndersonJ23115-TetR-double terminator(C3-C4 ) with: HindIII (C3-C4) , Expected bands : 2847 + 836 bp ,Positive result : C3,C4</i>]]
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[[File:T--Thessaly--Anderson--TETR-LEVEL1.png|700px|thumb|none|<i><b>Fig.2:</b>(U=Uncut C=Cut) Restriction digestion of T: AndersonJ23115-TetR-double terminator(C3-C4 ) with: HindIII (C3-C4) , Expected bands : 2847 + 836 bp ,Positive result : C3,C4</i>]]
  
  

Revision as of 00:41, 28 October 2020


pAndersonJ23115:RBS:tetR-terminator



Usage and Biology

This parts consists of AndersonJ23115 promoter BBa_K3505012 , TetR BBa_K3505005 and Double Terminator BBa_K3505017. By this way ,as AndersonJ23115 is a strong constitutive promoter, TetR is constitutively expressed. TetR binds to tetracycline response element and inhibits the expression of a desirable transcription unit (Gossen and Bujard, 1993).

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 cloned in alpha1R vector BBa_K3505008 and has overhangs compatible for GoldenBraid cloning.

Figure 1. The level A module of Tet-Off system : AndersonJ23115:RBS-TetR-Double terminator

Verification of Cloning

Fig.2:(U=Uncut C=Cut) Restriction digestion of T: AndersonJ23115-TetR-double terminator(C3-C4 ) with: HindIII (C3-C4) , Expected bands : 2847 + 836 bp ,Positive result : C3,C4


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 76
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 11
    Illegal NheI site found at 34
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 76
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 76
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  • Gossen, M. and Bujard, H., 1993. Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. Nucleic Acids Research, 21(18), pp.4411-4412.