Basic Vector Information
pQCascade_crRNA-nt 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:
- pQCascade_crRNA-nt
- Antibiotic Resistance:
- Streptomycin
- Length:
- 8445 bp
- Type:
- pET & Duet Vectors (Novagen)
- Replication origin:
- CloDF13 ori
- 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
pQCascade_crRNA-nt 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 ℃.
pQCascade_crRNA-nt vector Sequence
LOCUS 40924_35908 8445 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 8445)
AUTHORS xsan
TITLE Direct Submission
REFERENCE 2 (bases 1 to 8445)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8445
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter complement(1..78)
/label=lacI promoter
promoter 124..142
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 143..167
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
RBS 182..204
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
promoter 411..429
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 430..454
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
CDS 497..1678
/codon_start=1
/label=TniQ(WP_000479715.1)
/translation="MFLQRPKPYSDESLESFFIRVANKNGYGDVHRFLEATKRFLQDID
HNGYQTFPTDITRINPYSAKNSSSARTASFLKLAQLTFNEPPELLGLAINRTNMKYSPS
TSAVVRGAEVFPRSLLRTHSIPCCPLCLRENGYASYLWHFQGYEYCHSHNVPLITTCSC
GKEFDYRVSGLKGICCKCKEPITLTSRENGHEAACTVSNWLAGHESKPLPNLPKSYRWG
LVHWWMGIKDSEFDHFSFVQFFSNWPRSFHSIIEDEVEFNLEHAVVSTSELRLKDLLGR
LFFGSIRLPERNLQHNIILGELLCYLENRLWQDKGLIANLKMNALEATVMLNCSLDQIA
SMVEQRILKPNRKSKPNSPLDVTDYLFHFGDIFCLWLAEFQSDEFNRSFYVSRW"
CDS 1682..3604
/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 3576..4634
/codon_start=1
/label=Cas7
/note="accession number: WP_000775488.1"
/translation="MKLPTNLAYERSIDPSDVCFFVVWPDDRKTPLTYNSRTLLGQMEA
ASLAYDVSGQPIKSATAEALAQGNPHQVDFCHVPYGASHIECSFSVSFSSELRQPYKCN
SSKVKQTLVQLVELYETKIGWTELATRYLMNICNGKWLWKNTRKAYCWNIVLTPWPWNG
EKVGFEDIRTNYTSRQDFKNNKNWSAIVEMIKTAFSSTDGLAIFEVRATLHLPTNAMVR
PSQVFTEKESGSKSKSKTQNSRVFQSTTIDGERSPILGAFKTGAAIATIDDWYPEATEP
LRVGRFGVHREDVTCYRHPSTGKDFFSILQQAEHYIEVLSANKTPAQETINDMHFLMAN
LIKGGMFQHKGD"
CDS 4868..5236
/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 5266..5313
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(5487..6275)
/codon_start=1
/label=SmR
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
promoter complement(6276..6367)
/label=AmpR promoter
rep_origin complement(6415..7153)
/direction=LEFT
/label=CloDF13 ori
/note="Plasmids containing the CloDF13 (CDF) origin of
replication can be propagated in E. coli cells that contain
additional plasmids with compatible origins."
protein_bind complement(7329..7350)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(7366..8445)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
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