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 Vector Map
Plasmid Resuspension 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 ℃.
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"
This page is informational only.