Difference between revisions of "Part:BBa K4370011:Design"

 
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===Design Notes===
 
===Design Notes===
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This sequence has been designed to target different strains such as <i> S. ambofaciens </i> ATCC 23877, <i> S. bottropensis </i> ATCC 25435, <i> S. venezuelae </i> ATCC 10712 and <i> S. rimosus </i> ATCC 10970. The part is also designed to be cloned between NcoI and SnaBI sites into the pCRISPR-dCas9 plasmid published by Tong and collaborators (ACS Synthetic Biology, 2015). This plasmid encodes a dead version of Cas9 that can be used to silence gene expression at specific loci. Twenty nucl. (ACCTGAACCTTCTGTGCCAC) are perfect the beginning of lsr2A gene, the following nucleotide. (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCT) correspond to the gRNA scaffold.
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This sequence has been designed to target <i> lsr2A </i> gene in different strains such as <i> S. ambofaciens </i> ATCC 23877, <i> S. bottropensis </i> ATCC 25435, <i> S. venezuelae </i> ATCC 10712 and <i> S. rimosus </i> ATCC 10970. The part is also designed to be cloned between NcoI (CCATGG) and SnaBI (TACGTA) sites into the pCRISPR-dCas9 plasmid published by Tong and collaborators (ACS Synthetic Biology, 2015). This plasmid encodes a dead version of Cas9 that can be used to silence gene expression at specific loci. Twenty nucl. (ACCTGAACCTTCTGTGCCAC) are perfect the beginning of lsr2A gene, the following nucleotide. (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCT) correspond to the gRNA scaffold.
  
 
Please have a look to our CRISPRi tuto to learn more about the way to design sgRNA for gene expression silencing in bacteria.
 
Please have a look to our CRISPRi tuto to learn more about the way to design sgRNA for gene expression silencing in bacteria.
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===Source===
 
===Source===
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===References===
 
===References===
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Yaojun Tong, Pep Charusanti, Lixin Zhan, Tilmann Weber, and Sang Yup Lee, “CRISPR-Cas9 Based Engineering of Actinomycetal Genomes”, ACS Synth. Biol. 2015, 4, 9, 1020–1029
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https://doi.org/10.1021/acssynbio.5b00038
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Designed using our CRISPRi tuto.

Latest revision as of 08:09, 22 August 2022


sgRNA_lsr2A_STREPTO


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


Design Notes

This sequence has been designed to target lsr2A gene in different strains such as S. ambofaciens ATCC 23877, S. bottropensis ATCC 25435, S. venezuelae ATCC 10712 and S. rimosus ATCC 10970. The part is also designed to be cloned between NcoI (CCATGG) and SnaBI (TACGTA) sites into the pCRISPR-dCas9 plasmid published by Tong and collaborators (ACS Synthetic Biology, 2015). This plasmid encodes a dead version of Cas9 that can be used to silence gene expression at specific loci. Twenty nucl. (ACCTGAACCTTCTGTGCCAC) are perfect the beginning of lsr2A gene, the following nucleotide. (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCT) correspond to the gRNA scaffold.

Please have a look to our CRISPRi tuto to learn more about the way to design sgRNA for gene expression silencing in bacteria.

Source

The sequence has been synthetized.

References

Yaojun Tong, Pep Charusanti, Lixin Zhan, Tilmann Weber, and Sang Yup Lee, “CRISPR-Cas9 Based Engineering of Actinomycetal Genomes”, ACS Synth. Biol. 2015, 4, 9, 1020–1029

https://doi.org/10.1021/acssynbio.5b00038

Designed using our CRISPRi tuto.