Gene/Insert name FnCpf1 (humanized) Alt name type V CRISPR-associated protein Cpf1 (humanized) Alt name Cpf1 (humanized) Alt name Francisella tularensis subsp. novicida U112 Cpf1 (humanized) Species Francisella tularensis subsp. novicida U112 Insert Size (bp) 3910 Promoter T7 Tags / Fusion Proteins 6xHis (N terminal on backbone) MBP (N terminal on insert) TEV site (N terminal on insert) Nucleoplasmin NLS (C terminal on insert) 3xHA (C terminal on insert)
| 载体基本信息 | |||
| 载体名称 | 6-His-MBP-TEV-FnCpf1 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 10685 bp | 载体类型 | CRISPR Plasmids |
| 载体来源 | Feng Zhang | 拷贝数 | High copy number |
| 启动子 | T7 | 克隆方法 | Enzyme Cut |
| 5引物 | T7 promoter | 3引物 | T7 terminator |
| 载体标签 | MBP | 表达方法 | IPTG induced |
6-His-MBP-TEV-FnCpf1 载体载体图谱
6-His-MBP-TEV-FnCpf1 载体载体序列
LOCUS Exported 10685 bp ds-DNA circular SYN 04-JUL-2018
DEFINITION Bacterial expression plasmid for protein purification.
ACCESSION .
VERSION .
KEYWORDS 6-His-MBP-TEV-FnCpf1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10685)
AUTHORS Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS,
Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin
EV, Zhang F
TITLE Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas
System.
JOURNAL Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi:
10.1016/j.cell.2015.09.038.
PUBMED 26422227
REFERENCE 2 (bases 1 to 10685)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..10685
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 532..1120
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 1306..1445
/note="bom"
/note="basis of mobility region from pBR322"
CDS complement(1547..1738)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(2547..3629)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(3630..3707)
/gene="lacI"
/note="lacI promoter"
promoter 4016..4034
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 4035..4059
/bound_moiety="lac repressor encoded by lacI"
/note="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 4074..4096
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 4104..9326
/codon_start=1
/note="6His-MBP-TEV-FnCpf1-NLS-3xHA"
/translation="MGHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTV
EHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDA
VRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY
FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAE
AAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNK
ELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPN
IPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGASTGSLVPRGSASENLYFQGMSIY
QEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIE
EILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNL
FNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKG
FHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE
ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKG
INEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQ
IAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAV
LEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFE
EILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNN
LLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDE
KFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEG
YKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEF
NIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISE
SYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAEL
FYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFK
SSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDR
MKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFED
LNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKK
MGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFE
FSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIE
YGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDS
RQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNKR
PAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA"
CDS 4110..4127
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
regulatory 5082..5091
/regulatory_class="other"
/note="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 5244..5261
/codon_start=1
/product="thrombin recognition and cleavage site"
/note="thrombin site"
/translation="LVPRGS"
CDS 5268..5288
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/note="TEV site"
/translation="ENLYFQG"
CDS 9189..9236
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 9243..9323
/codon_start=1
/product="three tandem HA epitope tags"
/note="3xHA"
/translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
CDS 9348..9371
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/note="FLAG"
/translation="DYKDDDDK"
CDS 9372..9416
/codon_start=1
/product="peptide tag that allows for enzymatic
biotinylation"
/note="AviTag(TM)"
/translation="GLNDIFEAQKIEWHE"
CDS 9432..9449
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 9516..9563
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 9600..10055
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10081..10185
/gene="bla"
/note="AmpR promoter"
CDS 10186..361
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
