Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017475 | Nme2sgRNA_pLKO | 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:
- Nme2sgRNA_pLKO
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9023 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro
- Promoter:
- hPGK
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
Nme2sgRNA_pLKO 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.
Nme2sgRNA_pLKO vector Sequence
LOCUS V017475 9023 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V017475
VERSION V017475
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 9023)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9023
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin complement(168..756)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(930..1787)
/label="AmpR"
/note="beta-lactamase"
promoter complement(1788..1892)
/label="AmpR promoter"
rep_origin complement(1918..2373)
/direction=LEFT
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 2515..2531
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 2541..2559
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin complement(2580..2715)
/direction=LEFT
/label="SV40 ori"
/note="SV40 origin of replication"
polyA_signal complement(2742..2876)
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
LTR complement(2948..3181)
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
CDS complement(3312..3908)
/label="PuroR"
/note="puromycin N-acetyltransferase"
promoter complement(3930..4440)
/label="hPGK promoter"
/note="human phosphoglycerate kinase 1 promoter"
misc_feature complement(4489..4606)
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
misc_RNA complement(4655..4747)
/label="Nm tracrRNA"
/note="trans-activating CRISPR RNA for the Neisseria
meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
CDS 4940..5731
/label="NeoR/KanR"
/note="aminoglycoside phosphotransferase"
promoter complement(5873..6113)
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
CDS complement(6221..6262)
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
CDS complement(6411..6455)
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
misc_feature complement(6640..6873)
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature complement(7366..7491)
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
LTR complement(7538..7718)
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
promoter complement(7719..7945)
/label="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
promoter complement(7973..7991)
/label="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(8012..8028)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(8036..8052)
/label="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(8060..8090)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
mobile_element 8090..8857
/label="IS1"
/note="prokaryotic transposable element"
protein_bind complement(8882..8903)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."