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)

Replication origin:: ori

Source/Author:: Sanne E. Klompe, Phuc L. H. Vo, Tyler S. Halpin-Healy & Samuel H. Sternberg

Copy Number:: High copy number

Promoter:: tet

Cloning Method:: Enzyme Cut

5' Primer:: T7 promoter

3' Primer:: T7 terminator

Expression Method:: IPTG induced

His-MBP-Cas8_Cas7_Cas6 vector Map

Copy Sequence View Map

His-MBP-Cas8_Cas7_Cas6 vector Sequence

LOCUS       40924_1304        9524 bp 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
REFERENCE   2  (bases 1 to 9524)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        104..208
                     /label=AmpR promoter
     CDS             209..1066
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1240..1828
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(2014..2156)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2261..2449)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        4010..4028
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4117..4139
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4159..4185
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     CDS             4198..5298
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     CDS             5353..5373
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage 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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