From 2014.igem.org
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| - | Background on the construction: some flow this summer showed that pdCas9 with crRNA targeting this GFP1 sequence repressed nicely (as did GFP3 but not GFP2) so we picked this one to try making decoys for. The actual sequence is the 20bp of GFP1, the PAM, and then 10 more bp of spacer that come from the GFP orf. We added the spacer reasoning that dCas9 occupies space outside of just the 20bp matching crRNA, and after eyeballing the crystal structure 4008.pdb (which should be referenced & pictured) it looked like we needed about enough space for 1 additional turn of DNA outside of the 20bp. 10.5bp is ~1 turn, 3 bp is taken care of by PAM, so we added 10 to be safe. MZ was involved in this design stuff | + | <h2> Construction Background </h2> |
| - | | + | Some flow this summer showed that pdCas9 with crRNA targeting this GFP1 sequence repressed nicely (as did GFP3 but not GFP2) so we picked this one to try making decoys for. The actual sequence is the 20bp of GFP1, the PAM, and then 10 more bp of spacer that come from the GFP orf. We added the spacer reasoning that dCas9 occupies space outside of just the 20bp matching crRNA, and after eyeballing the crystal structure 4008.pdb (which should be referenced & pictured) it looked like we needed about enough space for 1 additional turn of DNA outside of the 20bp. 10.5bp is ~1 turn, 3 bp is taken care of by PAM, so we added 10 to be safe. |
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| | <groupparts>iGEM013 Duke</groupparts> | | <groupparts>iGEM013 Duke</groupparts> |
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Revision as of 15:22, 16 October 2014
Construction Background
Some flow this summer showed that pdCas9 with crRNA targeting this GFP1 sequence repressed nicely (as did GFP3 but not GFP2) so we picked this one to try making decoys for. The actual sequence is the 20bp of GFP1, the PAM, and then 10 more bp of spacer that come from the GFP orf. We added the spacer reasoning that dCas9 occupies space outside of just the 20bp matching crRNA, and after eyeballing the crystal structure 4008.pdb (which should be referenced & pictured) it looked like we needed about enough space for 1 additional turn of DNA outside of the 20bp. 10.5bp is ~1 turn, 3 bp is taken care of by PAM, so we added 10 to be safe.
iGEM013 Duke
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