Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001185 | pCAG-enAsCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nuc)-3xHA (AAS1815) | 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:
- pCAG-enAsCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nuc)-3xHA (AAS1815)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9176 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- CAG
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- pCAG-F (5'-GCAACGTGCTGGTTATTGTG-3')
- 3' Primer:
- Bglob-pA-R (5'-ttttggcagagggaaaaaga-3')
pCAG-enAsCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nuc)-3xHA (AAS1815) 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.
pCAG-enAsCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nuc)-3xHA (AAS1815) vector Sequence
LOCUS 40924_8016 9176 bp DNA circular SYN 13-MAY-2021
DEFINITION Mammalian expression plasmid for human codon optimized
enAsCas12a-HF1 (enhanced high-fidelity AsCas12a) encoding
E174R/N282A/S542R/K548R substitutions.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9176)
AUTHORS Kleinstiver BP, Sousa AA, Walton RT, Tak YE, Hsu JY, Clement K,
Welch MM, Horng JE, Malagon-Lopez J, Scarfo I, Maus MV, Pinello L,
Aryee MJ, Joung JK
TITLE Engineered CRISPR-Cas12a variants with increased activities and
improved targeting ranges for gene, epigenetic and base editing.
JOURNAL Nat Biotechnol. 2019 Feb 11. pii: 10.1038/s41587-018-0011-0. doi:
10.1038/s41587-018-0011-0.
PUBMED 30742127
REFERENCE 2 (bases 1 to 9176)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9176)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2019 Feb 11. pii: 10.1038/s41587-018-0011-0. doi:
10.1038/s41587-018-0011-0."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9176
/mol_type="other DNA"
/organism="synthetic DNA construct"
intron 288..1304
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 1312..1331
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
CDS 1403..5323
/codon_start=1
/label=AsCpf1
/note="CRISPR-associated protein from Acidaminococcus sp.
BV3L6 (Zetsche et al., 2015)"
/translation="MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND
HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN
AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD
KFTTYFSGFYRNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHF
ENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL
ALAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL
RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG
KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL
KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA
RNYATKKPYSVEKFKLNFQMPTLARGWDVNREKNNGAILFVKNGLYYLGIMPKQKGRYK
ALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP
LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSID
LSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG
HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLK
DQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFF
HVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR
SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV
VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ
FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD
VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN
HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM
RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES
KDLKLQNGISNQDWLAYIQELRN"
CDS 5324..5371
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 5378..5458
/codon_start=1
/product="three tandem HA epitope tags"
/label=3xHA
/translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
primer_bind complement(5548..5567)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 5613..5668
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(5667..5686)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(6029..6045)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6053..6069)
/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(6077..6107)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(6122..6143)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6201..6397
/label=SV40 promoter
/note="SV40 early promoter"
polyA_signal 6403..6537
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(6605..6622)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6776..7364)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7538..8395)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(8396..8500)
/label=AmpR promoter
enhancer 8531..8910
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"