Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000759 | pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) | 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:
- pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9258 bp
- Type:
- Mammalian Expression, CRISPR
- Replication origin:
- ori
- Promoter:
- EF-1α
pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) 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.
pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) vector Sequence
LOCUS 40924_40427 9258 bp DNA circular SYN 13-MAY-2021
DEFINITION This plasmid contains expression cassette for NmCas9 with N and C
NLS and an HA tag, a cassette for expression of tracrRNA, a cassette
for cloning crRNA under the control of U6 promoter..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9258)
AUTHORS Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ,
Thomson JA
TITLE Efficient genome engineering in human pluripotent stem cells using
Cas9 from Neisseria meningitidis.
JOURNAL Proc Natl Acad Sci U S A. 2013 Aug 12.
PUBMED 23940360
REFERENCE 2 (bases 1 to 9258)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9258)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl
Acad Sci U S A. 2013 Aug 12."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9258
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter complement(44..284)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 322..340
/label=T7 Term
/note="T7 terminator, reverse primer"
promoter complement(359..377)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_RNA complement(419..511)
/label=Nm tracrRNA
/note="trans-activating CRISPR RNA for the Neisseria
meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
promoter complement(519..759)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(824..843)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
promoter 993..2166
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
regulatory 2172..2181
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 2184..2204
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 2202..5447
/codon_start=1
/label=NmCas9
/note="Cas9 endonuclease from the Neisseria meningitidis
Type II CRISPR/Cas system (Esvelt et al., 2013)"
/translation="VAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRV
FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIK
SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEF
GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE
KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE
GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
CDS 5448..5474
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
CDS 5481..5507
/codon_start=1
/label=NLS
/note="nuclear localization signal (Makkerh et al., 1996)"
/translation="PAAKKKKLD"
polyA_signal 5529..5753
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 5942..7055
/label=mini-oriP
/note="internally truncated variant of the Epstein-Barr
virus oriP replication origin (Tanaka et al., 1999)"
primer_bind complement(7101..7117)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(7125..7141)
/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(7149..7179)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(7194..7215)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(7332..7349)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(7503..8091)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8265..9122)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(9123..9227)
/label=AmpR promoter