Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007538 | Nme2Cas9_AAV | 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:
- Nme2Cas9_AAV
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7270 bp
- Type:
- Mammalian Expression, AAV
- Replication origin:
- ori
- Promoter:
- U6
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- GAGGATAAGCGGCCCGCAGCAA
- 3' Primer:
- TTTCTTTTTCTTGGCTTGACCTGCCTTC
Nme2Cas9_AAV 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.
Nme2Cas9_AAV vector Sequence
LOCUS 40924_2244 7270 bp DNA circular SYN 13-MAY-2021
DEFINITION All-in-one AAV plasmid expressing Nme2Cas9 with sgRNA cassette.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7270)
AUTHORS Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y,
Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ
TITLE A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo
Genome Editing.
JOURNAL Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi:
10.1016/j.molcel.2018.12.003.
PUBMED 30581144
REFERENCE 2 (bases 1 to 7270)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7270)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell.
2018 Dec 18. pii: S1097-2765(18)31033-5. doi:
10.1016/j.molcel.2018.12.003."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7270
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 1..21
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 28..75
/codon_start=1
/label=nucleoplasmin NLS
/note="bipartite nuclear localization signal from
nucleoplasmin"
/translation="KRPAATKKAGQAKKKK"
CDS 3328..3375
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 3376..3465
/codon_start=1
/label=3xHA
/note="three tandem HA epitope tags"
/translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
CDS 3472..3498
/codon_start=1
/label=NLS
/note="nuclear localization signal (Makkerh et al., 1996)"
/translation="PAAKKKKLD"
primer_bind complement(3504..3523)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 3569..3624
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
repeat_region 3637..3777
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 3852..4307
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4324..4343)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4443..4465
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4503..4521)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4589..4693
/label=AmpR promoter
CDS 4694..5551
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5725..6313
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
repeat_region 6375..6504
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
misc_RNA complement(6532..6624)
/label=Nm tracrRNA
/note="trans-activating CRISPR RNA for the Neisseria
meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
promoter complement(6683..6923)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
regulatory 7259..7268
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"