Part:BBa_K3505014

AndersonJ23115:TetO:RBS GB compatible with A1-B2

Constitutive Anderson BBa_J23115with Tet Operator and RBS BBa_B0034

Usage and Biology

This part is a constitutive promoter from Andesron family , J23115 , having downstream with a Τet Operator and RBS. The activity of this part is regulated by TetR BBa_K3505003. TetR binds to TRE -tetracycline response element, and inhibits the expression of the desirable transcription unit. This part can be used in a Tet-off system, expression of Tet-regulated genes can be inhibited by tetracycline and its chemical derivatives , such as anhydrotetracycline aTc-a strong chemical repressor , that binds to TRE and the desirable transcription unit is blocked.

Design Notes

The sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 BBa_K3505007 and has overhangs compatible for Golden Braid cloning. This has position A1-B3.

Fig.1:The overhangs of this part in the Golden Braid Grammar

Figure 2. The level 0 module : pupd2- AndersonJ23115:TetO:RBS (illustration from SnapGene)

Verification of cloning

Fig.1:Figure 7: (U=Uncut , C= Cut) Restriction digestion of TetO (C1-C4 ) with :Not-HF(C1-C4) , Expected bands : 2046 + 129 bp, Positive result: C1,C2,C4

Experimental Use and Experience

This part is used in BBa_K3505034

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 11
  Illegal NheI site found at 34
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]