Difference between revisions of "Part:BBa K3407007:Design"

 
 
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===Design Notes===
 
===Design Notes===
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The target sequence (protospacer) is a sequence of 20 nucleotides that must precede a PAM sequence (NGG).
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This biobrick represents the DNA template for the transcription of a 65 nt shRNA, excluding the promoter. Trancription was performed with <html><a id="6" href="https://parts.igem.org/Part:BBa_K3407023" target="_blank"><b>BBa_K3407023</b></a></html>. The dsRNA sequence was taken from pUC57-OriLR-deGFP plasmid (BBa_K3407006). It corresponds to 27nt from the eGFP gene (nt 78 to 105), and is the one recognised and processed by Dicer to form siRNA. The shRNA has a single-stranded RNA (ssRNA) mutated loop region designed to deplete interaction with Fox-1 RBD (<html><a id="6" href="https://parts.igem.org/Part:BBa_K3407004" target="_blank"><b>BBa_K3407004</b></a></html>) <html><a href="#1">[1]</a></html>, containing its C3U and G6A mutations “<html><u>UG<b>U</b>AU<b>A</b>U</u><html>” in Fox-1 RBD's RNA target sequence. The dsRNA region contains a GG overhang that would represent the overhang left when mini-3 (<html><a id="6" href="https://parts.igem.org/Part:BBa_K3407002" target="_blank"><b>BBa_K3407002</b></a></html>) cleaves a tshRNA (See <html><a id="6" href="https://2020.igem.org/Team:TUDelft" target="_blank"><b>iGEM TU Delft 2020</b></a></html>, Design - Future perspectives), and that represents a good substrate to process the shRNA by Dicer into a siRNA.
  
  
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===References===
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<html>
  
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===Source===
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<head>
  
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Synthetically synthesised
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<title>Ordered List</title>
  
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===References===
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</head>
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<style>
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ol li {
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counter-increment: OrderedList;
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list-style: none;
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}
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ol li :before {
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content: "[" counter(OrderedList) "]";
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}
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</style>
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<html>
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<ol>
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<li>
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<a id="1" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1356361/" target="_blank">
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Auweter, S., Fasan, R., Reymond, L., Underwood, J., Black, D., Pitsch, S. and Allain, F., 2020. Molecular Basis Of RNA Recognition By The Human Alternative Splicing Factor Fox-1.</a>
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</li>
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</ol>
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</html>

Latest revision as of 10:31, 27 October 2020


shRNA*: short hairpin RNA with loop mutations.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This biobrick represents the DNA template for the transcription of a 65 nt shRNA, excluding the promoter. Trancription was performed with BBa_K3407023. The dsRNA sequence was taken from pUC57-OriLR-deGFP plasmid (BBa_K3407006). It corresponds to 27nt from the eGFP gene (nt 78 to 105), and is the one recognised and processed by Dicer to form siRNA. The shRNA has a single-stranded RNA (ssRNA) mutated loop region designed to deplete interaction with Fox-1 RBD (BBa_K3407004) [1], containing its C3U and G6A mutations “UGUAUAU” in Fox-1 RBD's RNA target sequence. The dsRNA region contains a GG overhang that would represent the overhang left when mini-3 (BBa_K3407002) cleaves a tshRNA (See iGEM TU Delft 2020, Design - Future perspectives), and that represents a good substrate to process the shRNA by Dicer into a siRNA.


References

Ordered List

  1. Auweter, S., Fasan, R., Reymond, L., Underwood, J., Black, D., Pitsch, S. and Allain, F., 2020. Molecular Basis Of RNA Recognition By The Human Alternative Splicing Factor Fox-1.