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
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
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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