载体基本信息 | |||
载体名称 | pMBP-LbCas12a | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 9653 bp | 载体类型 | Bacterial Expression |
拷贝数 | High Copy | 启动子 | T7 |
克隆方法 | Gibson Cloning | 5'引物 | GATGAAGCCCTGAAAGACGCGCAG |
3'引物 | CTA GTT ATT GCT CAG CGG T |
pMBP-LbCas12a 载体载体图谱
pMBP-LbCas12a 载体载体序列
LOCUS Exported 9653 bp ds-DNA circular SYN 08-JUN-2021 DEFINITION Expresses 10xHis-MBP fused to LbCas12a. ACCESSION . VERSION . KEYWORDS pMBP-LbCas12a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9653) AUTHORS Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA TITLE CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. JOURNAL Science. 2018 Apr 27;360(6387):436-439. doi: 10.1126/science.aar6245. Epub 2018 Feb 15. PUBMED 29449511 REFERENCE 2 (bases 1 to 9653) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1126/science.aar6245"; journalName: "Science"; date: "2018-04-27- 27"; volume: "360"; issue: "6387"; pages: "436-439" FEATURES Location/Qualifiers source 1..9653 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 990..1009 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1093..1112 /label=T7 /note="T7 promoter, forward primer" promoter 1093..1111 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 1200..1222 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1242..1271 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 1281..2381 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" primer_bind 2355..2378 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 2436..2456 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 2463..6146 /codon_start=1 /product="CRISPR-associated protein from Lachnospiraceae bacterium ND2006 (Zetsche et al., 2015)" /label=LbCpf1 /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFS EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI NTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ QGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRN YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS VKH" primer_bind complement(6275..6293) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6289..6336 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(6702..6730) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(6757..6775) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6843..6947 /gene="bla" /label=AmpR promoter CDS 6948..7808 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(7166..7185) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7979..8567 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8468..8487 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8721..8738 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8753..8892 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(8818..8837) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8978..9000 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8994..9185) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL"
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