Difference between revisions of "Part:BBa K3791004:Design"
 
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===Design Notes===
 
===Design Notes===
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For choosing which fragment of the spectinomycin-resistant gene we used for our spacer design, we had to take into account the PAM sequence needed by the nature of LbCas12a. In our case, the PAM sequence was TTTV, where V is any nucleotide except T. The PAM must be placed in the 5' extreme of the complementary chain of the one being targeted and must not appear in the target sequence. [1]
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For choosing which fragment of the spectinomycin-resistant gene we used for our spacer design, we had to take into account the PAM sequence needed by the nature of LbCas12a. In our case, the PAM sequence was TTTV, where V is any nucleotide except T. The PAM must be placed in the 5' extreme of the complementary chain of the one being targeted and must not appear in the target sequence [2].
  
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Another important consideration to bear in mind was that the spacer length was required to be between 18 and 24 nucleotides. [1]
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Another important consideration to bear in mind was that the spacer length was required to be between 18 and 24 nucleotides [2].
  
  
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===References===
 
===References===
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[1] New England Biolabs. (s/f). <b>How do I design a guide RNA for use with EnGen Lba Cas12a?</b> Neb.com. From: https://international.neb.com/faqs/2018/05/03/how-do-i-design-a-guide-rna-for-use-with-engen-lba-cas12a
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[1] Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O., & Zhang, F. (2019). SHERLOCK: nucleic acid detection with CRISPR nucleases. Nature Protocols, 14(10), 2986–3012. https://doi.org/10.1038/s41596-019-0210-2
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[2] New England Biolabs. (s/f). <b>How do I design a guide RNA for use with EnGen Lba Cas12a?</b> Neb.com. From: https://international.neb.com/faqs/2018/05/03/how-do-i-design-a-guide-rna-for-use-with-engen-lba-cas12a

Latest revision as of 22:19, 21 October 2021


Spacer gRNA Spectinomycin


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

For choosing which fragment of the spectinomycin-resistant gene we used for our spacer design, we had to take into account the PAM sequence needed by the nature of LbCas12a. In our case, the PAM sequence was TTTV, where V is any nucleotide except T. The PAM must be placed in the 5' extreme of the complementary chain of the one being targeted and must not appear in the target sequence [2].

Another important consideration to bear in mind was that the spacer length was required to be between 18 and 24 nucleotides [2].


Source

This part is the complementary sequence of a spectinomycin-resistant gene DNA fragment.

References

[1] Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O., & Zhang, F. (2019). SHERLOCK: nucleic acid detection with CRISPR nucleases. Nature Protocols, 14(10), 2986–3012. https://doi.org/10.1038/s41596-019-0210-2

[2] New England Biolabs. (s/f). How do I design a guide RNA for use with EnGen Lba Cas12a? Neb.com. From: https://international.neb.com/faqs/2018/05/03/how-do-i-design-a-guide-rna-for-use-with-engen-lba-cas12a