Part:BBa_K5102079:Design
pRAM_syntheticUTR-ProgRAM-recording-tape2.0
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 18
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 18
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2451
Illegal BamHI site found at 3511
Illegal XhoI site found at 2520 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 18
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 18
Illegal NgoMIV site found at 4856
Illegal AgeI site found at 3602 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
The composite part consists of: CMV enhancer, CMV promotor, 5' synthetic UTR, T7 promoter, recording tape 1.0, 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.
Source
The composite was assembled from gblocks and oligos provided by a DNA synthesis provider.
References
1. Castillo-Hair, S. et al. Optimizing 5’UTRs for mRNA-delivered gene editing using deep learning. Nat Commun 15, 5284 (2024). 2. Truong, D.-J. J. et al. Exonuclease-enhanced prime editors. Nat Methods 21, 455–464 (2024).