His-MBP-Cas8_Cas7_Cas6 vector (V001462)

Basic Vector Information

His-MBP-Cas8_Cas7_Cas6 expression plasmid used in paper: Klompe SE, Vo PLH, Halpin-Healy TS, Sternberg SH. Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature. 2019

      • Vector Name:
      • His-MBP-Cas8_Cas7_Cas6
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 9524 bp
      • Type:
      • pET & Duet Vectors (Novagen)
      • Source/Author:
      • Sanne E. Klompe, Phuc L. H. Vo, Tyler S. Halpin-Healy & Samuel H. Sternberg
      • Copy Number:
      • High copy number
      • Promoter:
      • T7
      • Cloning Method:
      • Enzyme Cut
      • 5' Primer:
      • T7 promoter
      • 3' Primer:
      • T7 terminator
      • Expression Method:
      • IPTG induced

His-MBP-Cas8_Cas7_Cas6 vector Vector Map

His-MBP-Cas8_Cas7_Cas69524 bp400800120016002000240028003200360040004400480052005600600064006800720076008000840088009200AmpR promoterAmpRoribomropT7 promoterRBS10xHisMBPTEV siteCas8Cas6T7 terminatortet promoter

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 ℃.

His-MBP-Cas8_Cas7_Cas6 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                9524 bp ds-DNA     circular SYN 15-JUN-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9524)
  AUTHORS   xsan
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9524
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             209..1069
                     /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"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2014..2156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2258..2449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        4010..4028
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4117..4139
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4159..4188
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4198..5298
                     /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"
     CDS             5353..5373
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5380..7302
                     /codon_start=1
                     /label=Cas8
                     /note="accession number:  WP_001194012.1"
                     /translation="MQTLKELIASNPDDLTTELKRAFRPLTPHIAIDGNELDALTILVN
                     LTDKTDDQKDLLDRAKCKQKLRDEKWWASCINCVNYRQSHNPKFPDIRSEGVIRTQALG
                     ELPSFLLSSSKIPPYHWSYSHDSKYVNKSAFLTNEFCWDGEISCLGELLKDADHPLWNT
                     LKKLGCSQKTCKAMAKQLADITLTTINVTLAPNYLTQISLPDSDTSYISLSPVASLSMQ
                     SHFHQRLQDENRHSAITRFSRTTNMGVTAMTCGGAFRMLKSGAKFSSPPHHRLNSKRSW
                     LTSEHVQSLKQYQRLNKSLIPENSRIALRRKYKIELQNMVRSWFAMQDHTLDSNILIQH
                     LNHDLSYLGATKRFAYDPAMTKLFTELLKRELSNSINNGEQHTNGSFLVLPNIRVCGAT
                     ALSSPVTVGIPSLTAFFGFVHAFERNINRTTSSFRVESFAICVHQLHVEKRGLTAEFVE
                     KGDGTISAPATRDDWQCDVVFSLILNTNFAQHIDQDTLVTSLPKRLARGSAKIAIDDFK
                     HINSFSTLETAIESLPIEAGRWLSLYAQSNNNLSDLLAAMTEDHQLMASCVGYHLLEEP
                     KDKPNSLRGYKHAIAECIIGLINSITFSSETDPNTIFWSLKNYQNYLVVQPRSINDETT
                     DKSSL"
     CDS             7274..8332
                     /codon_start=1
                     /label=Cas7
                     /note="accession number: WP_000775488.1"
                     /translation="MKLPTNLAYERSIDPSDVCFFVVWPDDRKTPLTYNSRTLLGQMEA
                     ASLAYDVSGQPIKSATAEALAQGNPHQVDFCHVPYGASHIECSFSVSFSSELRQPYKCN
                     SSKVKQTLVQLVELYETKIGWTELATRYLMNICNGKWLWKNTRKAYCWNIVLTPWPWNG
                     EKVGFEDIRTNYTSRQDFKNNKNWSAIVEMIKTAFSSTDGLAIFEVRATLHLPTNAMVR
                     PSQVFTEKESGSKSKSKTQNSRVFQSTTIDGERSPILGAFKTGAAIATIDDWYPEATEP
                     LRVGRFGVHREDVTCYRHPSTGKDFFSILQQAEHYIEVLSANKTPAQETINDMHFLMAN
                     LIKGGMFQHKGD"
     CDS             8566..8934
                     /codon_start=1
                     /product="type I-F CRISPR-associated endoribonuclease 
                     Cas6/Csy4 [Vibrio cholerae]"
                     /label=Cas6
                     /note="accession number:  WP_000872476.1"
                     /translation="MEQLQYFHISNTVLVPEDCTYVSFRRCQSIDKLTAAGLARKIRRL
                     EKRALSRGEQFDPSSFAQKEHTAIAHYHSLGESSKQTNRNFRLNIRMLSEQPREGNSIF
                     SSYGLSNSENSFQPVPLI"
     terminator      9074..9121
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(9487..9515)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"

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