Part:BBa_K2643000:Design

dxCas9-linker-Tn5 fusion protein (HIS Tag)

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3823
  Illegal PstI site found at 244
  Illegal PstI site found at 2212
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  Illegal PstI site found at 3962
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3823
  Illegal PstI site found at 244
  Illegal PstI site found at 2212
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  Illegal PstI site found at 3962
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3823
  Illegal BglII site found at 251
  Illegal BglII site found at 1188
  Illegal BamHI site found at 2006
  Illegal XhoI site found at 3544
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3823
  Illegal PstI site found at 244
  Illegal PstI site found at 2212
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  Illegal PstI site found at 3962
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3823
  Illegal PstI site found at 244
  Illegal PstI site found at 2212
  Illegal PstI site found at 2446
  Illegal PstI site found at 3658
  Illegal PstI site found at 3962
  Illegal NgoMIV site found at 4139
  Illegal NgoMIV site found at 5582
  Illegal NgoMIV site found at 5607
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2886
  Illegal BsaI site found at 3740
  Illegal BsaI.rc site found at 1411
  

Design Notes

The pSB1C3 backbone and dxCas9-linker-Tn5 were combined with the use of Gibson assembly. Subsequently, the Gibson assembly product was transformed into chemical competent E.coli DH5α cells using heat shock via the [http://2018.igem.org/Team:TUDelft/Experiments#chemcomptcellsprep-scroll chemical competent cell transformation protocol]. The transformation resulted in white colonies.

Source