Basic Vector Information
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | CRISPRoff-v2.1 | Antibiotic Resistance | Ampicillin |
| Length | 11885 bp | Type | Mammalian Expression |
| Copy Number | High Copy | Promoter | CAG |
| Cloning Method | Gibson Cloning | 5' Primer | ggcaaagaattctgcagtcg |
| 3' Primer | ccaccaccttctgataggc | ||
CRISPRoff-v2.1 vector Vector Map
Plasmid Resuspension 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 ℃.
CRISPRoff-v2.1 vector Sequence
LOCUS Exported 11885 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Expresses CRISPRoff-v2.1
(DNMT3A-DNMT3L-XTEN80-dCas9-HA-2xNLS-BFP-KRAB) downstream of the CAG
promoter.
ACCESSION .
VERSION .
KEYWORDS CRISPRoff-v2.1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11885)
AUTHORS Nunez JK, Chen J, Pommier GC, Cogan JZ, Replogle JM, Adriaens C,
Ramadoss GN, Shi Q, Hung KL, Samelson AJ, Pogson AN, Kim JYS, Chung
A, Leonetti MD, Chang HY, Kampmann M, Bernstein BE, Hovestadt V,
Gilbert LA, Weissman JS
TITLE Genome-wide programmable transcriptional memory by CRISPR-based
epigenome editing.
JOURNAL Cell. 2021 Apr 7. pii: S0092-8674(21)00353-6. doi:
10.1016/j.cell.2021.03.025.
PUBMED 33838111
REFERENCE 2 (bases 1 to 11885)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2021
Apr 7. pii: S0092-8674(21)00353-6. doi: 10.1016/j.cell.2021.03.025."
FEATURES Location/Qualifiers
source 1..11885
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 5..384
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 386..662
/label=chicken beta-actin promoter
intron 663..1679
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 1603..1625
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 1687..1706
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
CDS 3650..7753
/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="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 7757..7783
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 7802..7822
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7829..7849
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7889..8584
/codon_start=1
/product="monomeric blue fluorescent protein"
/label=TagBFP
/note="mammalian codon-optimized"
/translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
LPSKLGHKLN"
CDS 8630..8815
/codon_start=1
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
/label=KRAB
/translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
QLTKPDVILRLEKGEEP"
primer_bind complement(8907..8926)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 8972..9027
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(9026..9045)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(9388..9404)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9388..9404)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(9401..9423)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9412..9428
/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(9436..9466)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9481..9502
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9561..9756
/label=SV40 promoter
/note="SV40 early promoter"
rep_origin 9607..9742
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 9669..9688
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
polyA_signal 9762..9896
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9799..9818)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9853..9872
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(9964..9981)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(10135..10723)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(10215..10234)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10894..11754)
/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 11517..11536
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(11755..11859)
/gene="bla"
/label=AmpR promoter
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