Part:BBa_K5102075:Design

pRAM_ProgRAM-recording-tape3.0

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 18
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 18
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2494
  Illegal BamHI site found at 3554
  Illegal XhoI site found at 2563
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 18
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 18
  Illegal NgoMIV site found at 4899
  Illegal AgeI site found at 3645
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The composite part consists of: CMV enhancer, CMV promotor, T7 promoter, recording tape 3.0, T2A, miRFP670nano3, P2A, eUnaG, T2A, mScarlet3, P2A, eUnaG, T2A, mTagBFP2, P2A, eUnaG, human beta-globin 3'UTR, PP7, T7 terminator, WPRE, SV40 polyA. 5' CMV UTR, human beta-globin 3' UTR, and WPRE elements were added to the part to increase mRNA expression and stability. All the fluorescent proteins have been codon optimized for expression in mammalian cells in all three forward open reading frames. miRFPnano3, mScarlet3 and mTagBFP2 are used as an indicator of the current state of the tape. eUnaG is included to provide a translational-level control of total protein expression throughout the system. PP7 has been added to the part to increase the binding of the editor fused with tdPCP protein and therefore increase the editing efficiency.

<img src="" style="background-color: transparent; width: 100px ;display: block; margin: 0 auto;"/>

Source

The composite was assembled from gblocks and oligos provided by a DNA synthesis provider.

References

Truong, D.-J. J. et al. Exonuclease-enhanced prime editors. Nat Methods 21, 455–464 (2024).