Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Josephina Hendrix
Date created: 2014-09-24 11:00:00
Date modified: 2015-05-08 01:10:27
Endogenous Type II CRISPR-Cas9 phagemid
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K1445001_sequence (Version 1) |
Description
The M13ori-pCas9 composite part consists of the M13 origin of replication (M13ori) and the pCas9 part (BBa_K1218011) containing a tracrRNA sequence, the cas9 gene, and a minimal CRISPR array.M13ori (BBa_K1445000) is a 500bp noncoding sequence that is recognized by the gene II of the M13 phage, which results in the uptake of the plasmid containing the ori into an assembled phage coat. This part does not contain any complete phage genes so phage production requires a Helper Phagemid such as M13K07. There will be some packaging of Helper Phagemid but their packaging signal weakened so parts containing the M13ori are preferentially packaged.
pCas9 contains the native trcrRNA and promoter region upstream of the Cas9 protein. The type II Cas9 protein is the native, active version from streptococcus pyogenes with the exceptrion of a SNP to erase an illegal EcoRI cleavage site. Downstream of the Cas9 protein is a minimal CRISPR array. This minimal array only includes two CRISPR repeats flanking a spacer region. It is this spacer region that determines which DNA sequence is targeted by the Cas9 endonuclease. Spacer sequence can be easily replaced with the protocol found here.
Notes
We have experienced some toxic effects by the pCas9 part so we suggest growing part-containing cells in lower concentrations of antibiotic, even if plasmid contains a high copy origin of replication.Source
The M13 origin of replication from Litmus28i and part BBa_K1218011 from the registry.| Access | Instance | Definition |
| public public | BBa_K1445000 BBa_K1218011 | BBa_K1445000 BBa_K1218011 |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_K1445000 BBa_K1218011 | 1,529 538,5617 | BBa_K1445000 BBa_K1218011 |