Composite

Part:BBa_K3505033

Designed by: Asteria Tsapadikou   Group: iGEM20_Thessaly   (2020-10-20)
Revision as of 16:45, 27 October 2020 by Asteriat (Talk | contribs) (→‎Usage and Biology)


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 can bind to tetracycline response element and inhibits the expression of a desirable transcription unit

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 Golden Braid cloning.

Figure 2. The level A module of Tet-Off system : AndersonJ23115:RBS-TetR-Double terminator


Verification of Cloning

Fig.1:(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]
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Categories
Parameters
None