| 出品公司: | ZYbscience |
|---|---|
| 载体名称: | pET-Cas9-NLS-6xHis |
| 质粒类型: | CRISPR/Cas9载体；细菌基因敲除载体 |
| 高拷贝/低拷贝: | 高拷贝 |
| 插入位点: | 限制性内切酶，多克隆位点 |
| 启动子: | lac/T7 |
| 载体大小: | 9265 bp |
| 5' 测序引物及序列: | T7 forward：5’TAATACGACTCACTATAGGG 3’ |
| 3' 测序引物及序列: | T7 Terminal：5’GCTAGTTATTGCTCAGCGG 3’ |
| 载体标签: | NLS 、 His tag (C-ter） |
| 载体抗性: | Ampicillin |
| 克隆菌株: | Mach1 |
| 宿主细胞（系）: | 细菌，如大肠杆菌 |
| 备注: | |
| 产品目录号: | #62933 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 诱导型 |
| 病毒/非病毒: | 非病毒 |
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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20℃低温保存
- 保质期：
三年
- 英文名：
pET-Cas9-NLS-6xHis
- 库存：
20
- 供应商：
泽叶生物
载体基本信息
载体质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS Exported 9265 bp ds-DNA circular SYN 26-JUL-2016
DEFINITION Bacterial vector for regulated expression of S. pyogenes Cas9 with a
C-terminal nuclear localization signal and 6xHis tag.
ACCESSION .
VERSION .
KEYWORDS pET-Cas9-NLS-6xHis
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9265)
AUTHORS Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder
ML, Joung JK, Chen ZY, Liu DR.
TITLE Cationic lipid-mediated delivery of proteins enables efficient
protein-based genome editing in vitro and in vivo.
JOURNAL Nat. Biotechnol. 2015;33:73-80.
PUBMED 25357182
REFERENCE 2 (bases 1 to 9265)
AUTHORS Liu Lab / Addgene #62933
TITLE Direct Submission
JOURNAL Exported Tuesday, July 26, 2016 from SnapGene Viewer 3.1.4
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..9265
/organism="synthetic DNA construct"
/lab_host="Escherichia coli"
/mol_type="other DNA"
protein_bind 3..27
/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 42..64
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 71..4174
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/note="Cas9"
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 4175..4195
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 4205..4222
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 4307..4354
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 4391..4846
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(4961..5821)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5822..5914)
/gene="bla"
/note="AmpR promoter"
rep_origin 5995..6583
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7013..7204)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(7776..8858)
/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(8859..8936)
/gene="lacI"
/note="lacI promoter"
promoter 9249..2
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
61 gagatatacc atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg
121 atgggcggtg atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa
181 tacagaccgc cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga
241 gacagcggaa gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa
301 tcgtatttgt tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt
361 ctttcatcga cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc
421 tatttttgga aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca
481 tctgcgaaaa aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc
541 cttagcgcat atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga
601 taatagtgat gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga
661 agaaaaccct attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag
721 taaatcaaga cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt
781 atttgggaat ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga
841 tttggcagaa gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa
901 tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc
961 agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct
1021 atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc
1081 tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa
1141 cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa
1201 accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga
1261 tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg
1321 tgagctgcat gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg
1381 tgagaagatt gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg
1441 tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa
1501 ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa
1561 ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta
1621 ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc
1681 agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg
1741 aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag
1801 tgttgaaatt tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt
1861 gctaaaaatt attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga
1921 ggatattgtt ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa
1981 aacatatgct cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac
2041 tggttgggga cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa
2101 aacaatatta gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat
2161 ccatgatgat agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg
2221 cgatagttta catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat
2281 tttacagact gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga
2341 aaatatcgtt attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc
2401 gcgagagcgt atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa
2461 agagcatcct gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca
2521 aaatggaaga gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga
2581 tgtcgatcac attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt
2641 aacgcgttct gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa
2701 aaagatgaaa aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt
2761 tgataattta acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat
2821 caaacgccaa ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag
2881 tcgcatgaat actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac
2941 cttaaaatct aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga
3001 gattaacaat taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt
3061 gattaagaaa tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga
3121 tgttcgtaaa atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt
3181 cttttactct aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat
3241 tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg
3301 gcgagatttt gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa
3361 aacagaagta cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga
3421 caagcttatt gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc
3481 aacggtagct tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt
3541 aaaatccgtt aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa
3601 tccgattgac tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa
3661 actacctaaa tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc
3721 cggagaatta caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata
3781 tttagctagt cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt
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CRISPR/Cpf1 系统，并利用该系统简单、高效地实现了水稻多基因定点编辑。CRISPR/Cpf1 与 CRISPR/Cas9 一样，都是目前最为简捷、高效和最易实现多基因编辑的基因组编辑技术。两者表达载体的构建都相当简单，且都只需要串联多个 crRNA 即可实现多位点编辑，而编辑效率和特异性也都相当，另因都受 PAM 识别位点的限制，编辑范围也都有一定的局限性。因此，CRISPR/Cpf1 与 CRISPR/Cas9 可以说是不相伯仲的基因组编辑技术，相当于 CRISPR/Cas 技术 “厨房
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蛋白封闭转录时，CAP对该系统不能发挥作用；而没有CAP存在时，即使没有阻遏蛋白与操纵序列结合，操纵子仍无转录活性。只有在CAP存在且没有阻遏蛋白与操纵序列结合时，或者说只有高乳糖低葡萄糖时，操纵子发挥最大转录活性。这种协调与细菌对碳源的优先利用相一致。 本实验使用的表达载体p32aGFPuv上含有乳糖操纵子的调节序列，目的基因表达的是带6XHis（组氨酸标签）的重组绿色荧光蛋白。在IPTG的诱导下，融合蛋白表达可增强105倍，并且可用金属鳌合亲和层析分离纯化，最终获得纯的重组绿色荧光蛋白。
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pET-Cas9-NLS-6xHis载体
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