Basic Vector Information
LbCpf1-2NLS 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 ℃.
LbCpf1-2NLS vector Sequence
LOCUS 40924_1609 10514 bp DNA circular SYN 13-MAY-2021 DEFINITION bacterial expression of LbCpf1-2NLS. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10514) AUTHORS Moreno-Mateos MA, Fernandez JP, Rouet R, Vejnar CE, Lane MA, Mis E, Khokha MK, Doudna JA, Giraldez AJ TITLE CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. JOURNAL Nat Commun. 2017 Dec 8;8(1):2024. doi: 10.1038/s41467-017-01836-2. PUBMED 29222508 REFERENCE 2 (bases 1 to 10514) TITLE Direct Submission REFERENCE 3 (bases 1 to 10514) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat Commun."; date: "2017-12-8"; pages: " 10.1038/s41467-017-01836-2" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..10514 /mol_type="other DNA" /organism="synthetic DNA construct" primer_bind 506..525 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 609..627 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 716..738 /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 758..775 /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" CDS 785..1885 /codon_start=1 /label=MBP /note="maltose binding protein from E. coli" /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" CDS 1940..1960 /codon_start=1 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" /translation="ENLYFQS" CDS 1970..5653 /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 5657..5683 /codon_start=1 /label=HA /note="HA (human influenza hemagglutinin) epitope tag" /translation="YPYDVPDYA" CDS 5702..5722 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 5729..5749 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 5789..6493 /codon_start=1 /label=mCherry /note="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" CDS 6500..6520 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" terminator 6666..6713 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(7079..7107) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(7134..7152) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7220..7324 /label=AmpR promoter CDS 7325..8182 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 8356..8944 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9098..9115 /label=L4440 /note="L4440 vector, forward primer" misc_feature complement(9130..9269) /label=bom /note="basis of mobility region from pBR322" primer_bind 9355..9377 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(9374..9562) /codon_start=1 /label=rop /note="Rop protein, which maintains plasmids at low copy number" /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL"
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