Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Peivand Sadat Mousavi
Date created: 2015-09-17 11:00:00
Date modified: 2015-09-21 11:50:48
SgRNA targeting LacI promoter
| Types | DnaRegion |
| Roles | mature_transcript_region RNA |
| Sequences | BBa_K1645998_sequence (Version 1) |
Description
This part contains the 20 nucleotides long sequence which is complimentary to the region within the LacI (BBa_R0010) promoter, the scaffold region which is responsible for the secondary structure of the SgRNA, and the terminator. A constitutive promoter can should be placed upstream of this SgRNA in order for it to get transcribed.The suggested promoter to be used for this part is the U6 promoter.Notes
This part was cloned without the promoter to avoid the biobrick illegal site existing on the U6 promoter. You can refer Waterloo iGEM 2015 wiki to see the full sequence of the U6 promoter in order to clone it in. The characterization data using this part in our purposes suggests that our CRISPR cas9 system is working using this SgRNA by suppressing RFP which uses lacI promoter. Our experiment was done in BL21 and the SgRNA was in pSB3K3 backbone containing part BBa-K1645999. We were able to detect drop in RFP levels using this SgRNA coupled by S.pyrogenes dCas9.Source
The scaffold sequence was obtained from gRNA design protocol from Church Lab. Link is provided below:https://www.addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf