Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V004036 | pPlatTET-gRNA2 | 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:
- pPlatTET-gRNA2
- Antibiotic Resistance:
- Kanamycin
- Length:
- 14283 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Morita S, Noguchi H, Horii T, Nakabayashi K, Kimura M, Okamura K, Sakai A, Nakashima H, Hata K, Nakashima K, Hatada I.
- Promoter:
- SV40
pPlatTET-gRNA2 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.
pPlatTET-gRNA2 vector Sequence
LOCUS 40924_34929 14283 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pPlatTET-gRNA2 DNA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14283)
AUTHORS Morita S, Noguchi H, Horii T, Nakabayashi K, Kimura M, Okamura K,
Sakai A, Nakashima H, Hata K, Nakashima K, Hatada I.
TITLE Targeted DNA demethylation in vivo using dCas9-peptide repeat and
scFv-TET1 catalytic domain fusions
JOURNAL Nat. Biotechnol. 34 (10), 1060-1065 (2016)
PUBMED 27571369
REFERENCE 2 (bases 1 to 14283)
AUTHORS Hatada I, Morita S.
TITLE Direct Submission
JOURNAL Submitted (19-JUL-2016) Contact:Izuho Hatada Gunma University,
Institute for Molecular and Cellular Regulation; 3-39-15
Showa-machi, Maebashi, Gunma 371-8512, Japan URL
:http://epigenome.dept.showa.gunma-u.ac.jp/~hatada/
REFERENCE 3 (bases 1 to 14283)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 14283)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat.
Biotechnol."; date: "2016"; volume: "34"; issue: "10"; pages:
"1060-1065"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(19-JUL-2016) Contact:Izuho Hatada Gunma University, Institute for
Molecular and Cellular Regulation"; volume: " 3-39-15 Showa-machi,
Maebashi, Gunma 371-8512, Japan URL :http"; pages:
"//epigenome.dept.showa.gunma-u.ac.jp/~hatada"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14283
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 61..364
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 367..642
/label=chicken beta-actin promoter
intron 643..1651
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
CDS 1727..1747
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 1802..5905
/codon_start=1
/label=dCas9
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 5909..5935
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 5954..5974
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5981..6001
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6065..6121
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label=GCN4_v4
/translation="EELLSKNYHLENEVARLKK"
CDS 6188..6244
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label=GCN4_v4
/translation="EELLSKNYHLENEVARLKK"
CDS 6311..6367
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label=GCN4_v4
/translation="EELLSKNYHLENEVARLKK"
CDS 6434..6490
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label=GCN4_v4
/translation="EELLSKNYHLENEVARLKK"
CDS 6557..6613
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label=GCN4_v4
/translation="EELLSKNYHLENEVARLKK"
CDS 6701..6757
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 7505..7531
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
CDS 7622..8332
/codon_start=1
/label=superfolder GFP
/note="GFP variant that folds robustly even when fused to
poorly folded proteins (Nager et al., 2011)"
/translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
VTAAGITHGMDELYK"
promoter 9994..11704
/label=CAG
/note="CMV early enhancer fused to modified chicken
beta-actin promoter"
promoter 11694..11785
/label=AmpR promoter
promoter 11787..12144
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 12179..12970
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 13205..13252
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 13581..14169
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"