Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017384 | pEvolvR-enCas9-PolI5M | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pEvolvR-enCas9-PolI5M
- Antibiotic Resistance:
- Kanamycin
- Length:
- 12546 bp
- Replication origin:
- ori
- Promoter:
- tetR/tetAs
pEvolvR-enCas9-PolI5M vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
pEvolvR-enCas9-PolI5M vector Sequence
LOCUS V017384 12546 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V017384
VERSION V017384
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 12546)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..12546
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 116..171
/label="tetR/tetA promoters"
/note="overlapping promoters for bacterial tetR and tetA"
CDS 207..4310
/label="eSpCas9(1.1)"
/note="Cas9 endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system, mutated to improve targeting
specificity (Slaymaker et al., 2016)"
CDS 4380..7160
/gene="polA"
/label="DNA polymerase I"
/note="DNA polymerase I from Escherichia coli (strain K12).
Accession#: P00582"
CDS complement(7306..8118)
/label="KanR"
/note="aminoglycoside phosphotransferase"
rep_origin 8347..8935
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(9121..9260)
/label="bom"
/note="basis of mobility region from pBR322"
CDS complement(9365..9553)
/label="rop"
/note="Rop protein, which maintains plasmids at low copy
number"
CDS 10678..11391
/label="superfolder GFP"
/note="GFP variant that folds robustly even when fused to
poorly folded proteins (Pedelacq et al., 2006)"
terminator 11403..11474
/label="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 11490..11517
/label="T7Te terminator"
/note="phage T7 early transcription terminator"
misc_RNA 11531..11606
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 11627..11656
/label="Myc"
/note="Myc (human c-Myc proto-oncogene) epitope tag"
CDS 11672..11689
/label="6xHis"
/note="6xHis affinity tag"
terminator complement(11918..11961)
/label="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
CDS complement(join(12023..12546,1..100))
/label="TetR"
/note="tetracycline repressor TetR"