Part:BBa_K5102079
pRAM_syntheticUTR-ProgRAM-recording-tape2.0
The composite part is designed for the ProgRAM molecular recording system and features a recording tape composed of a series of START codons arranged in three forward open reading frames (ORFs). In the composite part, the tape is in state 0, indicating that no molecular events have been recorded, and all adenosines in the START codons remain unmodified by dPspCas13b-ADAR2DD fusion. Downstream of the tape, a sequence of fluorescent proteins enables visualization of the current recorded state. The design incorporates sequentially modifiable adenosine sites within the RNA tape, creating a dynamic "traffic light" system that allows for precise in vivo monitoring of recording events without disrupting cellular functions. Each adenosine corresponds to a START codon; upon deamination, this modification disrupts the codon, shifting the open reading frame by one base pair and triggering the expression of one of three distinct fluorescent proteins: dark-red (mRFP670nano3), orange (mScarlet3), or blue (mTagBFP2). To ensure proper protein folding, each fluorescent protein is encoded downstream of the RNA tape and preceded by a 2A peptide, which promotes ribosomal skipping during translation. This design guarantees the efficient production of fluorescent proteins without interference from upstream sequences. Additionally, eUnaG (Truong et al., 2024), a small green fluorescent protein codon-optimized for all three ORFs, is included to provide a translational-level control of total protein expression throughout the system. The composite part also contains elements to enhance mRNA expression and stability, such as the 5' CMV UTR, human beta-globin 3' UTR, and WPRE.
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' synthetic UTR, human beta-globin 3' UTR, and WPRE elements were added to the part to increase mRNA expression and stability. The 5' synthetic UTR was designed utilizing the deep learning model developed by Castillo-Hair et al. (2024), which optimizes 5’ UTRs for efficient mRNA translation using generative neural networks and gradient descent. This model was trained on polysome profiling data from randomized 5’ UTR libraries across multiple cell types, allowing it to learn sequence features that enhance translation efficiency. The model was validated by calculation of the mean ribosome load (MRL) and minimum free energy (MFE) for each designed UTR.
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.
Sequence and Features
- 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]
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