Part:BBa_K3807029:Design

plant enCas9-PolI5M expression cassette

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 884
  Illegal EcoRI site found at 8924
  Illegal PstI site found at 7052
  Illegal PstI site found at 8504
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 884
  Illegal EcoRI site found at 8924
  Illegal PstI site found at 7052
  Illegal PstI site found at 8504
  Illegal NotI site found at 872
  Illegal NotI site found at 8912
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 884
  Illegal EcoRI site found at 8924
  Illegal BamHI site found at 5294
  Illegal BamHI site found at 7312
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 884
  Illegal EcoRI site found at 8924
  Illegal PstI site found at 7052
  Illegal PstI site found at 8504
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 884
  Illegal EcoRI site found at 8924
  Illegal PstI site found at 7052
  Illegal PstI site found at 8504
  Illegal NgoMIV site found at 2204
  Illegal NgoMIV site found at 6205
  Illegal AgeI site found at 5702
  Illegal AgeI site found at 7292
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 896
  Illegal SapI.rc site found at 2285
  Illegal SapI.rc site found at 8936
  

Design Notes

Assembled following the Golden Gate @ VIB manual using VIB gateway collection vectors. CamV35SP promoter and terminator were chosen because they are commonly used in plants strong regulators. NLS-N7 is used to guide the EvolvR into the nucleus. P2A-mCherry-NLS is used as transfromation marker as it is separated from the EvolvR and the independently is transported into the nucleus where red fluorescence can be detected.

Source

ViB gateway collection vectors #p00232, #p0100, #p00423, #p0195, #p01239, #p00339, Addgene plasmids #113077 and #113078

References

Halperin et al, 2018; https://gatewayvectors.vib.be/information