Basic Vector Information
TETv4 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 ℃.
TETv4 vector Sequence
LOCUS 40924_48927 11679 bp DNA circular SYN 13-MAY-2021 DEFINITION Expresses TETv4 (TET1CD-XTEN80-dCas9-3xNLS-P2A-BFP) downstream of the CAG promoter. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11679) 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 11679) TITLE Direct Submission REFERENCE 3 (bases 1 to 11679) 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." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..11679 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 5..384 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 386..661 /label=chicken beta-actin promoter primer_bind 1116..1138 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1200..1219 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" regulatory 1250..1259 /label=Kozak sequence /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" /regulatory_class="other" CDS 3656..7759 /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 7775..7795 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 7799..7819 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 7823..7843 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 7856..7912 /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 7913..8608 /codon_start=1 /label=TagBFP /note="monomeric blue fluorescent protein" /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD LPSKLGHKLN" primer_bind complement(8701..8720) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 8766..8821 /label=beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(8820..8839) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(9182..9198) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9182..9198) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(9195..9217) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 9206..9222 /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(9230..9260) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(9275..9296) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 9354..9550 /label=SV40 promoter /note="SV40 early promoter" polyA_signal 9556..9690 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(9758..9775) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(9929..10517) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(10691..11548) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(11549..11653) /label=AmpR promoter
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