Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001068 | pSQT1601 | 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:
- pSQT1601
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10323 bp
- Type:
- Mammalian Expression, CRISPR
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- CAG
- 5' Primer:
- pCAG-F
- 3' Primer:
- Bglob-pA-R
pSQT1601 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.
pSQT1601 vector Sequence
LOCUS Exported 10323 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION human codon optimized Csy4 and FokI-dCas9 expression plasmid.
ACCESSION .
VERSION .
KEYWORDS pSQT1601
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10323)
AUTHORS Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin
MJ, Aryee MJ, Joung JK
TITLE Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome
editing.
JOURNAL Nat Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908.
PUBMED 24770325
REFERENCE 2 (bases 1 to 10323)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908."
FEATURES Location/Qualifiers
source 1..10323
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(63..80)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(234..822)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(314..333)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(993..1853)
/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 1616..1635
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(1854..1958)
/gene="bla"
/label=AmpR promoter
enhancer 1989..2368
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
intron 2622..3638
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 3562..3584
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 3646..3665
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
regulatory 3731..3740
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4301..4354
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 4358..4378
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4385..4972
/codon_start=1
/product="nonspecific DNA cleavage domain of the FokI
endonuclease (Li et al., 1992)"
/label=FokI cleavage domain
/note="must dimerize to be catalytically active"
/translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
CDS 4988..9088
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 9095..9115
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 9122..9187
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
primer_bind complement(9277..9296)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 9342..9397
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(9396..9415)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(9758..9774)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9758..9774)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(9771..9793)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9782..9798
/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(9806..9836)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9851..9872
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9931..10126
/label=SV40 promoter
/note="SV40 early promoter"
rep_origin 9977..10112
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 10039..10058
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
polyA_signal 10132..10266
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(10169..10188)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 10223..10242
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"