Plasmid

Part:BBa_K4058031:Design

Designed by: Sarah Cumberland   Group: iGEM21_Guelph   (2021-10-20)


CLE18 multiplex gRNA in AlcR-pHSN6A0


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 10436
    Illegal XbaI site found at 5564
    Illegal SpeI site found at 12599
    Illegal PstI site found at 4635
    Illegal PstI site found at 5929
    Illegal PstI site found at 6475
    Illegal PstI site found at 7897
    Illegal PstI site found at 8101
    Illegal PstI site found at 9343
    Illegal PstI site found at 15507
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 10436
    Illegal NheI site found at 7141
    Illegal SpeI site found at 12599
    Illegal PstI site found at 4635
    Illegal PstI site found at 5929
    Illegal PstI site found at 6475
    Illegal PstI site found at 7897
    Illegal PstI site found at 8101
    Illegal PstI site found at 9343
    Illegal PstI site found at 15507
    Illegal NotI site found at 5556
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 10436
    Illegal BglII site found at 2550
    Illegal BglII site found at 6731
    Illegal BglII site found at 7421
    Illegal BamHI site found at 1554
    Illegal BamHI site found at 2292
    Illegal BamHI site found at 5533
    Illegal BamHI site found at 7025
    Illegal BamHI site found at 9063
    Illegal BamHI site found at 10160
    Illegal XhoI site found at 5525
    Illegal XhoI site found at 6349
    Illegal XhoI site found at 7531
    Illegal XhoI site found at 8695
    Illegal XhoI site found at 11260
    Illegal XhoI site found at 12354
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 10436
    Illegal XbaI site found at 5564
    Illegal SpeI site found at 12599
    Illegal PstI site found at 4635
    Illegal PstI site found at 5929
    Illegal PstI site found at 6475
    Illegal PstI site found at 7897
    Illegal PstI site found at 8101
    Illegal PstI site found at 9343
    Illegal PstI site found at 15507
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 10436
    Illegal XbaI site found at 5564
    Illegal SpeI site found at 12599
    Illegal PstI site found at 4635
    Illegal PstI site found at 5929
    Illegal PstI site found at 6475
    Illegal PstI site found at 7897
    Illegal PstI site found at 8101
    Illegal PstI site found at 9343
    Illegal PstI site found at 15507
    Illegal NgoMIV site found at 4060
    Illegal NgoMIV site found at 7293
    Illegal NgoMIV site found at 13336
    Illegal AgeI site found at 1258
    Illegal AgeI site found at 1386
    Illegal AgeI site found at 1917
    Illegal AgeI site found at 11633
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4349
    Illegal BsaI.rc site found at 3136
    Illegal SapI site found at 6267
    Illegal SapI.rc site found at 7734


Design Notes

This construct contains the AlcR promoter from the Caddick et. al (1998) with the addition of a translational enhancer extracted from the sequence for pHSN6A01. This promoter is inserted before the dCas9 gene in pHSN6A01 to put dCas9 under ethanol inducible control.

pHSN6A01 was a gift from Qi-Jun Chen (Addgene plasmid # 50586 ; http://n2t.net/addgene:50586 ; RRID:Addgene_50586)

The gRNA sequence was designed using the guide RNA design software CHOPCHOP. The parameters were set to find optimal CRISPR/Cas9 target sites for CRISPR activation. Target sequences were selected based on the predicted binding efficiency provided by CHOPCHOP.

Source

pHSN6A01 was a gift from Qi-Jun Chen (Addgene plasmid # 50586 ; http://n2t.net/addgene:50586 ; RRID:Addgene_50586)

The gRNA sequences come from the promoter sequence of the CLE18 gene found in Arabidopsis thaliana.

References

A CRISPR/Cas9 toolkit for multiplex genome editing in plants. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. BMC Plant Biol. 2014 Nov 29;14(1):327. 10.1186/s12870-014-0327-y PubMed 25432517

Caddick, M., Greenland, A., Jepson, l. et al. An ethanol inducible gene switch for plants used to manipulate carbon metabolism. Nat Biotechnol 16, 177–180 (1998). https://doi-org.subzero.lib.uoguelph.ca/10.1038/nbt0298-177

Labun, K., Montague, T. G., Krause, M., Torres Cleuren, Y. N., Tjeldnes, H., & Valen, E. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Research (2019).