Basic Vector Information
6His-MBP-TEV-huLbCpf1 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 ℃.
6His-MBP-TEV-huLbCpf1 vector Sequence
LOCUS 40924_110 10469 bp DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression plasmid for protein purification. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10469) AUTHORS Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F TITLE Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. JOURNAL Nat Biotechnol. 2017 Jan;35(1):31-34. doi: 10.1038/nbt.3737. Epub 2016 Dec 5. PUBMED 27918548 REFERENCE 2 (bases 1 to 10469) TITLE Direct Submission REFERENCE 3 (bases 1 to 10469) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt.3737"; journalName: "Nat Biotechnol"; date: "2017-01"; volume: "35"; issue: "1"; pages: "31-34" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..10469 /mol_type="other DNA" /organism="synthetic DNA construct" promoter complement(978..1055) /label=lacI promoter promoter 1364..1382 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1383..1407 /label=lac operator /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)." RBS 1422..1444 /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1458..1475 /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" CDS 1476..2573 /codon_start=1 /label=MBP /note="maltose binding protein from E. coli" /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQT" CDS 2592..2609 /codon_start=1 /label=thrombin site /note="thrombin recognition and cleavage site" /translation="LVPRGS" CDS 2616..2636 /codon_start=1 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" /translation="ENLYFQG" CDS 2637..6320 /codon_start=1 /label=LbCpf1 /note="CRISPR-associated protein from Lachnospiraceae bacterium ND2006 (Zetsche et al., 2015)" /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFS EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI NTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ QGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRN YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS VKH" CDS 6321..6368 /codon_start=1 /label=nucleoplasmin NLS /note="bipartite nuclear localization signal from nucleoplasmin" /translation="KRPAATKKAGQAKKKK" CDS 6375..6455 /codon_start=1 /label=3xHA /note="three tandem HA epitope tags" /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" CDS 6480..6503 /codon_start=1 /label=FLAG /note="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /translation="DYKDDDDK" CDS 6504..6548 /codon_start=1 /label=AviTag(TM) /note="peptide tag that allows for enzymatic biotinylation" /translation="GLNDIFEAQKIEWHE" CDS 6564..6581 /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" terminator 6648..6695 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 6732..7187 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 7213..7317 /label=AmpR promoter CDS 7318..8175 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 8349..8937 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature complement(9123..9262) /label=bom /note="basis of mobility region from pBR322" CDS complement(9367..9555) /codon_start=1 /label=rop /note="Rop protein, which maintains plasmids at low copy number" /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" protein_bind complement(10330..10351) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS complement(join(10367..10469,1..977)) /codon_start=1 /label=lacI /note="lac repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ"
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