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 Vector 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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