Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017246 | pHAGE-TO-nmdCas9-3XmCherry | 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:
- pHAGE-TO-nmdCas9-3XmCherry
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12104 bp
- Type:
- RNA interference
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Promoter:
- CMV/TO
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pHAGE-TO-nmdCas9-3XmCherry 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.
pHAGE-TO-nmdCas9-3XmCherry vector Sequence
LOCUS V017246 12104 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V017246
VERSION V017246
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 12104)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..12104
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..204
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 206..224
/label="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
CDS 384..404
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 417..3659
/label="NmdCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Neisseria meningitidis Type II CRISPR/Cas system"
CDS 3678..3698
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 3711..4418
/label="mCherry"
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
misc_feature 5892..6480
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 6555..6788
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
promoter 6993..7097
/label="AmpR promoter"
CDS 7098..7955
/label="AmpR"
/note="beta-lactamase"
rep_origin 8129..8717
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 9128..9149
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9164..9194
/label="lac promoter"
/note="promoter for the E. coli lac operon"
misc_feature 10183..10308
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 10805..11038
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 11223..11267
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 11416..11457
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 11560..11677
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
enhancer 11801..12104
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"