Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V008301 | pRH2502 | 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:
- pRH2502
- Antibiotic Resistance:
- Kanamycin
- Length:
- 8783 bp
- Type:
- Bacterial Expression, CRISPR
- Replication origin:
- ori
- Copy Number:
- Low Copy
- Promoter:
- uv15tetO
- Cloning Method:
- Restriction Enzyme
pRH2502 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.
pRH2502 vector Sequence
LOCUS V008301 8783 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V008301
VERSION V008301
KEYWORDS pRH2502
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8783)
AUTHORS Singh AK, Carette X, Potluri LP, Sharp JD, Xu R, Prisic S, Husson RN
TITLE Investigating essential gene function in Mycobacterium tuberculosis
using an efficient CRISPR interference system.
JOURNAL Nucleic Acids Res. 2016 Jul 12. pii: gkw625.
PUBMED 27407107
REFERENCE 2 (bases 1 to 8783)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8783)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
Acids Res. 2016 Jul 12. pii: gkw625."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8783
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 1..4104
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 4117..4137
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
polyA_signal 4158..4292
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(4488..5076)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5410..6222)
/label="KanR"
/note="aminoglycoside phosphotransferase"
CDS complement(6525..7637)
/gene="33"
/label="Integrase"
/note="Integrase from Mycobacterium phage L5. Accession#:
P22884"
terminator complement(8170..8216)
/label="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
regulatory 8405..8414
/label="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
protein_bind 8626..8643
/gene="tetO"
/label="tet operator"
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"