Part:BBa_K5102072
pRAM_ProgRAM-recording-tape1.0
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). 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: miRFP670nano3, mScarlet3, 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.
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 2494
Illegal BamHI site found at 3554
Illegal XhoI site found at 2563 - 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 4899
Illegal AgeI site found at 3645 - 1000COMPATIBLE WITH RFC[1000]
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