Lab Documentation
Part Collection and Characterization
1.0 Nine New Parts Submitted to the Registry
We designed and submitted a total of nine parts this year, forming the complete NeuroSplice detection system and providing necessary characterization controls. These parts range from basic regulatory elements (Riboswitch, Oligo) to the final composite reporter cassette.
BBa_25CPXDO0 (Toehold Riboswitch)
BBa_257KJT8O (Trigger Oligo)
BBa_25FW15AV (Composite Cassette)
BBa_25SIIXDB (sfGFP CDS)
BBa_25F7N9EO (Leader Insulator)
BBa_25EAUO3A (Trailer Insulator)
BBa_25MGETYD (Linker Peptide)
BBa_25NHXE3I (GG Buffer)
BBa_2563P5EX (Neg. Control Oligo)
1.1 Detailed Part Information
BBa_25CPXDO0: Toehold Riboswitch Sensing IL7R $\Delta 6$
Type
Riboswitch [SO:0000035]
Composition
Basic Part (5'UTR)
License
CC-BY 4.0
Designed By:
Shriya Anumarlu, et. al.
Source:
Designed by Team CalUCSF, iGEM 2025. Architecture based on toehold switches (Green et al., Cell, 2014, doi:10.1016/j.cell.2014.10.002). Targets the IL7R Delta6 splice isoform.
Functional Description
An RNA toehold riboswitch designed for translational activation. It is a molecular switch embedded in the 5′UTR of a reporter/CDS:
- OFF state: The RBS and AUG are sequestered in a stable stem-loop, blocking ribosome access.
- ON state: The cognate Trigger RNA (BBa_257KJT8O) binds, unfolds the hairpin, and exposes the RBS/start codon, allowing translation of the downstream protein.
Usage & Sequence Notes
This is a Basic Part (no promoter/terminator). Place directly upstream of the Coding Sequence (CDS).
Sequence: ACGATAGGCTTAATCCTGAGCTATTATTGAAACAGAGGAGATCAATAATGGCTCAGGAT
1.2 Characterization Trial Sequences
Below are the full DNA sequences used in the various optimization trials for the composite part, followed by the sequences for the active and control oligonucleotide triggers. Note that Trial 5 corresponds to the submitted part BBa_25FW15AV.
| Sequence Name | Nucleotide Sequence (5' to 3') |
|---|---|
| Trial 1 (amilCP no buffer) | taatacgactcactatagggacgataggcttaatcctgagctattattgaaacagaggagatcaataatggctcaggataacggtagcgcaggtagcggtcatagtgtgatcgctaaacaaatgacctacaaggtttatatgtcaggcacggtcaatggacactactttgaggtcgaaggcgatggaaaaggtaagccctacgagggggagcagacggtaaagctcactgtcaccaagggcggacctctgccatttgcttgggatattttatcaccacagtgtcagtacggaagcataccattcaccaagtaccctgaagacatccctgactatgtaaagcagtcattcccggagggctatacatgggagaggatcatgaactttgaagatggtgcagtgtgtactgtcagcaatgattccagcatccaaggcaactgtttcatctaccatgtcaagttctctggtttgaactttcctcccaatggacctgtcatgcagaagaagacacagggctgggaacccaacactgagcgtctctttgcacgagatggaatgctgctaggaaacaactttatggctctgaagttagaaggaggcggtcactatttgtgtgaatttaaaactacttacaaggcaaagaagcctgtgaagatgccagggtatcactatgttgaccgcaaactggatgtaaccaatcacaacaaggattacacttcggttgagcagtgtgaaatttccattgcacgcaaacctgtggtcgcctaaaaccccttggggcctctaaacgggtcttgaggggttttttg |
| Trial 2 (sfGFP no buffer) | taatacgactcactatagggacgataggcttaatcctgagctattattgaaacagaggagatcaataatggctcaggataacggtagcgcaggtagcggtcattacacacgaataaaagataacaaagatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggcgatgttaatgggcaaaaattctctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactgggaagctacctgttccatggccaacacttgtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatattttacaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaaatggaatacaactataactcacataatgtatacatcatggcagacaaaccaaagaatggaatcaaagttaacttcaaaattagacacaacattaaagatggaagcgttcaattagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttccaaagatcccaacgaaaagagagatcacatgatccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaatgtccagacttccaattgacactaaagtgtccgaacaattactaaattctcagggttcctggttaaattcaggctgagactttatttatatatttatagattcattaaaattttatgaataatttattgatgttattaataggggctattttcttattaaataggctactggagtgtataaccccttggggcctctaaacgggtcttgaggggttttttg |
| Trial 3 (sfGFP with buffer) | gagtgccagaaccgttattctcgatacccgctattaatcagagtggtggctctctccacgtaatacgactcactatagggacgataggcttaatcctgagctattattgaaacagaggagatcaataatggctcaggataacggtagcgcaggtagcggtcattacacacgaataaaagataacaaagatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggcgatgttaatgggcaaaaattctctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactgggaagctacctgttccatggccaacacttgtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatattttacaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaaatggaatacaactataactcacataatgtatacatcatggcagacaaaccaaagaatggaatcaaagttaacttcaaaattagacacaacattaaagatggaagcgttcaattagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttccaaagatcccaacgaaaagagagatcacatgatccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaatgtccagacttccaattgacactaaagtgtccgaacaattactaaattctcagggttcctggttaaattcaggctgagactttatttatatatttatagattcattaaaattttatgaataatttattgatgttattaataggggctattttcttattaaataggctactggagtgtataaccccttggggcctctaaacgggtcttgaggggttttttgaaatagtgtagggtggggctcgcgacaagattccgggaccggctgagggggagagcggcg |
| Trial 4 (sfGFP reduced G-rich) | gagtgccagaaccgttattctcgatacccgctattaatcagagtggtggctctctccacgtaatacgactcactatagggacgataggcttaatcctgagctattattgaaacagaggagatcaataatggctcaggataacggtagcgcaggtagcggtcattacacacgaataaaagataacaaagatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggcgatgttaatgggcaaaaattctctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactgggaagctacctgttccatggccaacacttgtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatattttacaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaaatggaatacaactataactcacataatgtatatacatcatggcagacaaaccaaagaatggaatcaaagttaacttcaaaattagacacaacattaaagatggaagcgttcaattagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttccaaagatcccaacgaaaagagagatcacatgatccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaatgtccagacttccaattgacactaaagtgtccgaacaattactaaattctcagggttcctggttaaattcaggctgagactttatttatatatttatagattcattaaaattttatgaataatttattgatgttattaataggggctattttcttattaaataggctactggagtgtataaccccttggggcctctaaacgggtcttgaggggttttttgaaatagtgtataataagactcgcgacaagattccggtaccggctgaccccgagagcggcg |
| Trial 5 (sfGFP to sgFP) / BBa_25FW15AV | GAGTGCCAGAACCGTTATTCTCGATACCCGCTATTAATCAGAGTGGTGGCTCTCTCCACGGGACGATAGGCTTAATCCTGAGCTATTATTGAAACAGAGGAGATCAATAATGGCTCAGGATAACGGTAGCGCAGGTAGCGGTCATAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGCGATGTTAATGGGCAAAAATTCTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGGAAGCTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTACAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAAATGGAATACAACTATAACTCACATAATGTATACATCATGGCAGACAAACCAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTAAAGATGGAAGCGTTCAATTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCCAAAGATCCCAACGAAAAGAGAGATCACATGATCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGAAATAGTGTATAATAAGACTCGCGACAAGATTCCGGTACCGGCTGACCCCGAGAGCGGCG |
| Oligonucleotide Triggers | |
| Oligo - sIL7R (DNA) | CAATAATAGCTCAGGATTAAGCCTATCG |
| Oligo - sIL7R (RNA) | CAAUAAUAGCUCAGGAUUAAGCCUAUCG |
| Control Oligo (DNA) | TGTGGCGCAGTATCATTACCGTGAGGCA |
| Control Oligo (RNA) | UGUGGCGCAGUAUCAUUACCGUGAGGCA |