Part:BBa_K3924053

ParaBAD-dCas9-linker-PmCDA1-terminator

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 311
  Illegal EcoRI site found at 1656
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 311
  Illegal EcoRI site found at 1656
  Illegal NheI site found at 300
  Illegal NheI site found at 1415
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 311
  Illegal EcoRI site found at 1656
  Illegal BglII site found at 5091
  Illegal BamHI site found at 239
  Illegal BamHI site found at 3694
  Illegal XhoI site found at 4700
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 311
  Illegal EcoRI site found at 1656
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 311
  Illegal EcoRI site found at 1656
  Illegal AgeI site found at 74
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5843
  Illegal SapI site found at 56
  

Profile

Name:ParaBAD-dCas9-linker-PmCDA1-terminator
Base Pairs: 5965bp
Origin: Escherichia coli
Properties: A mutanted Cas9 protein fused to a deaminase which can change C into U.A linker, terminator and a Arabinose induced induced promoter have been added

Usage and Biology

dCas9 can bind sgRNA and help CDA to target a specific site of target gene.The expression of dCas9 cen be induced by arabinose induced

Design and Construction

For this part, we can purchase directly. But to save time, we got it from Xing Lab, Tsinghua University.

Fig.1 Design for Target-AID part, the Target-AID protein is preceded by the ParaBAD (i.e. Arabinose promoter), which has very little leakage feature

Functional Verification

It has been reported functional ,but we failed to repeat it in our lab due to some reasons explained in Wiki.

Reference

[1]Banno S, Nishida K, Arazoe T, Mitsunobu H, Kondo A. Deaminase-mediated multiplex genome editing in Escherichia coli. Nat Microbiol. 2018;3(4):423-429. doi:10.1038/s41564-017-0102-6