Basic Vector Information
- Vector Name:
- 6His-MBP-TEV-huAsCpf1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10706 bp
- Type:
- Bacterial Expression
- Replication origin:
- ori
- Copy Number:
- Low Copy
6His-MBP-TEV-huAsCpf1 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 ℃.
6His-MBP-TEV-huAsCpf1 vector Sequence
LOCUS 40924_105 10706 bp DNA circular SYN 13-MAY-2021
DEFINITION Bacterial expression plasmid for protein purification.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10706)
AUTHORS Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J,
DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS,
Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F
TITLE Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.
JOURNAL Nat Biotechnol. 2017 Jan;35(1):31-34. doi: 10.1038/nbt.3737. Epub
2016 Dec 5.
PUBMED 27918548
REFERENCE 2 (bases 1 to 10706)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 10706)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt.3737";
journalName: "Nat Biotechnol"; date: "2017-01"; volume: "35"; issue:
"1"; pages: "31-34"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10706
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 538..559
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 1334..1522
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
misc_feature 1627..1766
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(1952..2540)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2714..3571)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(3572..3676)
/label=AmpR promoter
rep_origin complement(3702..4157)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
terminator complement(4194..4241)
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(4308..4325)
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS complement(4341..4385)
/codon_start=1
/label=AviTag(TM)
/note="peptide tag that allows for enzymatic biotinylation"
/translation="GLNDIFEAQKIEWHE"
CDS complement(4386..4409)
/codon_start=1
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/translation="DYKDDDDK"
CDS complement(4569..8489)
/codon_start=1
/label=AsCpf1
/note="CRISPR-associated protein from Acidaminococcus sp.
BV3L6 (Zetsche et al., 2015)"
/translation="MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND
HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN
AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD
KFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHF
ENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL
NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL
RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG
KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL
KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA
RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYK
ALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP
LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSID
LSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG
HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLK
DQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFF
HVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR
SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV
VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ
FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD
VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN
HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM
RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES
KDLKLQNGISNQDWLAYIQELRN"
CDS complement(8490..8510)
/codon_start=1
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
/translation="ENLYFQG"
CDS complement(8517..8534)
/codon_start=1
/label=thrombin site
/note="thrombin recognition and cleavage site"
/translation="LVPRGS"
CDS complement(8553..9650)
/codon_start=1
/label=MBP
/note="maltose binding protein from E. coli"
/translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
NYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFW
YAVRTAVINAASGRQTVDEALKDAQT"
CDS complement(9651..9668)
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
RBS complement(9682..9704)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(9719..9743)
/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)."
promoter complement(9744..9762)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 10071..10148
/label=lacI promoter
CDS join(10149..10706,1..522)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
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