Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000093 | pC015 - dLwCas13a-NF | 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:
- pC015 - dLwCas13a-NF
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10715 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- CAG
- 5' Primer:
- pCAG-F
pC015 - dLwCas13a-NF vector 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.
pC015 - dLwCas13a-NF vector Sequence
LOCUS Exported 10715 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Expresses negative-feedback dLwCas13a for mammalian RNA
targeting/imaging.
ACCESSION .
VERSION .
KEYWORDS pC015 - dLwCas13a-NF
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10715)
AUTHORS Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J,
Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES,
Voytas DF, Ting AY, Zhang F
TITLE RNA targeting with CRISPR-Cas13.
JOURNAL Nature. 2017 Oct 4. doi: 10.1038/nature24049.
PUBMED 28976959
REFERENCE 2 (bases 1 to 10715)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature.
2017 Oct 4. doi: 10.1038/nature24049."
FEATURES Location/Qualifiers
source 1..10715
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(281..1141)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 904..923
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(1142..1246)
/gene="bla"
/label=AmpR promoter
protein_bind 1297..1308
/label=ZF binding site
/bound_moiety="CCR5TC zinc-finger domain"
/note="target sequence for the CCR5TC zinc-finger domain
from the CCR5-224 (+) zinc-finger nuclease (Perez et al.,
2008; Gross et al., 2013)"
enhancer 1312..1691
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1694..1969
/label=chicken beta-actin promoter
intron 1970..2981
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 2905..2927
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 2989..3008
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
CDS 3060..6515
/codon_start=1
/product="catalytically dead mutant of a Leptotrichia wadei
CRISPR-Cas effector that acts as an RNA-guided RNAse
(Abudayyeh et al., 2017)"
/label=dLwCas13a
/note="contains the inactivating mutations R474A and
R1046A"
/translation="MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIY
IKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEY
DLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINEN
NVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEK
YKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLD
KEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLL
NKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETE
NENGITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNE
IEDFFANIDEAISSIAHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLK
IFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFW
SVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQ
KFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIE
SNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKY
TENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDF
ELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEK
IADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAI
GNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE
IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIANY
IAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKK
IEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE"
CDS 6561..7271
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELY"
CDS 6561..7271
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELY"
primer_bind complement(6603..6624)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(6864..6883)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 7211..7232
/label=EGFP-C
/note="EGFP, forward primer"
CDS 7317..7658
/codon_start=1
/product="zinc-finger domain from the CCR5-224 (+)
zinc-finger nuclease that recognizes the left half-site
target sequence in the human CCR5 gene (Perez et al., 2008;
Gross et al., 2013)"
/label=CCR5TC
/translation="FQCRICMRNFSDRSNLSRHIRTHTGEKPFACDICGRKFAISSNLN
SHTKIHTGSQKPFQCRICMRNFSRSDNLARHIRTHTGEKPFACDICGRKFATSGNLTRH
TKIHLRGSQL"
CDS 7707..7832
/codon_start=1
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the rat zinc finger protein Kid-1
(Witzgall et al., 1994)"
/label=KRAB-A
/translation="VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASM"
CDS 7842..7862
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
intron 7879..8011
/label=chimeric intron
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
primer_bind 7994..8013
/label=pMT2-F
/note="Synthetic intron, forward primer"
misc_feature 8020..8608
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8073..8093)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(8491..8502)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(8635..8652)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 8641..8752
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(8848..8868)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(8848..8866)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(8887..8903)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9009..9028)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 9074..9129
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(9128..9147)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(9490..9506)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 9514..9530
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(9538..9568)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9583..9604
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9663..9858
/label=SV40 promoter
/note="SV40 early promoter"
rep_origin 9709..9844
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 9771..9790
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
polyA_signal 9864..9998
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9901..9920)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9955..9974
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(10066..10083)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(join(10237..10715,1..110))
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(10317..10336)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"