| 质粒名称: | pEJS654 All-in-One AAV-sgRNA-hNmeCas9 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 112139 |
| 存储实验室: | Erik Sontheimer |
|
载体骨架基本信息 |
|
| 载体名称: | PX551-Plasmid #60957 |
| 载体出品公司: | Feng Zhang |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 2880 bp |
| 完整质粒大小: | 7278 bp |
| 载体类型: | 哺乳动物表达，小鼠靶向，AAV，CRISPR |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | sgRNA scaffold |
| 插入大小: | 121 bp |
| 启动子 | U6 |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | XbaI（销毁后未知） |
| 3' 克隆位点: | XbaI（销毁后未知） |
| 5' 测序引物 | ctctagaGTTTAAACAAA |
| 3' 测序引物 | GCATATACGATACAAGGCTGT |
|
文献引用方法 |
|
| 材料和方法部分: | pEJS654 All-in-One AAV-sgRNA-hNmeCas9 was a gift from Erik Sontheimer (Addgene plasmid # 112139 ; http://n2t.net/addgene:112139 ; RRID:Addgene_112139) |
| 参考文献部分: | All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo. Ibraheim R, Song CQ, Mir A, Amrani N, Xue W, Sontheimer EJ. Genome Biol. 2018 Sep 19;19(1):137. doi: 10.1186/s13059-018-1515-0. 10.1186/s13059-018-1515-0 PubMed 30231914 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pEJS654 All-in-One AAV-sgRNA-hNmeCas9 7267 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Delivery of human-codon-optimized Cas9 from Neisseria meningitidis
(NmeCas9) and its single-guide RNA in a single AAV vector for in
vivo genome editing..
ACCESSION .
VERSION .
KEYWORDS pEJS654 All-in-One AAV-sgRNA-hNmeCas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7267)
TITLE All-in-One Adeno-associated Virus Delivery and Genome Editing by
Neisseria meningitidis Cas9 in vivo
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 7267)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7267
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..130
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
misc_RNA complement(158..250)
/label=Nm tracrRNA
/note="trans-activating CRISPR RNA for the Neisseria
meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
promoter complement(309..549)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(359..378)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(529..549)
/label=hU6-F
/note="Human U6 promoter, forward primer"
regulatory 885..894
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 897..917
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 924..971
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 972..4214
/codon_start=1
/product="Cas9 endonuclease from the Neisseria meningitidis
Type II CRISPR/Cas system (Esvelt et al., 2013)"
/label=NmCas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="AAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVF
ERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKS
LPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAG
NAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFG
NPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTK
LNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDN
AEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE
EKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKE
IEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGR
LNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFK
ARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRV
FASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFD
GKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEK
LSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDL
EKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKT
GVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQL
IDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEG
IGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
CDS 4221..4268
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 4269..4358
/codon_start=1
/product="three tandem HA epitope tags"
/label=3xHA
/translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
CDS 4365..4391
/codon_start=1
/product="nuclear localization signal (Makkerh et al.,
1996)"
/label=NLS
/translation="PAAKKKKLD"
primer_bind complement(4397..4416)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_site 4462..4517
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal"
repeat_region 4530..4670
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4530..4659
/label=AAV2 ITR
rep_origin 4745..5200
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4832..4851)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5042..5063
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5217..5236)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5336..5358
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5396..5414)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5482..5586
/gene="bla"
/label=AmpR promoter
CDS 5587..6447
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(5805..5824)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6618..7206
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7107..7126
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
121 aggggttcct gcggcctcta gagtttaaac aaaaaaataa acgatgcccc ttaaagcaga
181 agctttaagg ggcagagcgt tgcggcacat cttttcagac ggccttattg tagcaacggt
241 tctcatttcg tttccgaaat gagaaaggga gctacaactg aagagctgga atgctcttcc
301 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
361 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
421 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
481 tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
541 taggccctct tcctgcccga ccttgacgtc gactctagaa tggaggcggt actatgtaga
601 tgagaattca ggagcaaact gggaaaagca actgcttcca aatatttgtg atttttacag
661 tgtagttttg gaaaaactct tagcctacca attcttctaa gtgttttaaa atgtgggagc
721 cagtacacat gaagttatag agtgttttaa tgaggcttaa atatttaccg taactatgaa
781 atgctacgca tatcatgctg ttcaggctcc gtggccacgc aactcatact taagcagaca
841 gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa caccgccacc atggtgccta
901 agaagaagag aaaggtggaa gataaacgcc cagcagctac aaagaaggca ggtcaagcca
961 agaaaaagaa agcagcattc aagccaaact caatcaatta catcctggga ctggacatcg
1021 gcatcgcatc cgtcgggtgg gctatggtcg aaatcgacga ggaggagaac cccatccgcc
1081 tgatcgatct gggcgtgcgc gtgtttgaga gggcagaggt gcctaagacc ggcgacagcc
1141 tggccatggc acggagactg gcacgctccg tgaggcgcct gacccggaga agggcccaca
1201 gactgctgag gacacgccgg ctgctgaaga gggagggcgt gctgcaggcc gccaacttcg
1261 atgagaatgg cctgatcaag tccctgccca ataccccttg gcagctgagg gcagccgccc
1321 tggaccgcaa gctgacacct ctggagtggt ccgccgtgct gctgcacctg atcaagcacc
1381 ggggctacct gtctcagaga aagaacgagg gcgagacagc cgataaggag ctgggcgccc
1441 tgctgaaggg agtggcagga aatgcacacg ccctgcagac cggcgacttt cgcacaccag
1501 ccgagctggc cctgaacaag ttcgagaagg agagcggcca catccgcaat cagcggtctg
1561 actatagcca caccttctcc cggaaggatc tgcaggccga gctgatcctg ctgtttgaga
1621 agcagaagga gttcggcaac ccacacgtgt ctggcggcct gaaggagggc atcgagacac
1681 tgctgatgac acagcggccc gccctgagcg gcgacgcagt gcagaagatg ctgggacact
1741 gcacctttga gccagccgag cccaaggccg ccaagaatac ctacacagcc gagcggttca
1801 tctggctgac aaagctgaac aatctgagga tcctggagca gggaagcgag cgcccactga
1861 ccgacacaga gagggccacc ctgatggatg agccctaccg caagtccaag ctgacatatg
1921 cacaggcaag gaagctgctg ggcctggagg acaccgcctt ctttaagggc ctgagatacg
1981 gcaaggataa cgccgaggcc tctacactga tggagatgaa ggcctatcac gccatcagca
2041 gggccctgga gaaggagggc ctgaaggaca agaagtcccc actgaatctg tctcccgagc
2101 tgcaggatga gatcggcacc gcctttagcc tgttcaagac cgacgaggat atcacaggca
2161 gactgaagga caggatccag ccagagatcc tggaggccct gctgaagcac atcagctttg
2221 ataagttcgt gcagatcagc ctgaaggccc tgcggaggat cgtgccactg atggagcagg
2281 gcaagaggta cgacgaggcc tgcgccgaaa tctacggcga tcactatggc aagaagaaca
2341 cagaggagaa aatctacctg ccccctatcc ccgccgatga gatcaggaac cctgtggtgc
2401 tgcgcgccct gtctcaggca agaaaagtga tcaacggagt ggtgcgccgg tacggcagcc
2461 ccgccagaat ccacatcgag acagccaggg aagtgggcaa gtcctttaag gacagaaagg
2521 agatcgagaa gaggcaggag gagaacagaa aggataggga gaaggccgcc gccaagttca
2581 gagagtactt tcctaatttc gtgggcgagc caaagtccaa ggatatcctg aagctgaggc
2641 tgtacgagca gcagcacggc aagtgtctgt attctggcaa ggagatcaac ctgggccgcc
2701 tgaatgagaa gggctatgtg gagatcgacc acgccctgcc tttttctcgg acctgggacg
2761 atagcttcaa caataaggtg ctggtgctgg gctctgagaa ccagaataag ggcaaccaga
2821 caccctacga gtatttcaac ggcaaggaca atagccgcga gtggcaggag tttaaggcaa
2881 gggtggagac aagcaggttc cctcggtcca agaagcagag aatcctgctg cagaagtttg
2941 acgaggatgg cttcaaggag aggaacctga atgacacccg ctacgtgaat cggtttctgt
3001 gccagttcgt ggccgataga atgaggctga ccggcaaggg caagaagaga gtgtttgcct
3061 ccaacggcca gatcacaaat ctgctgaggg gcttctgggg cctgagaaag gtgagggcag
3121 agaacgacag gcaccacgca ctggatgcag tggtggtggc atgttctacc gtggccatgc
3181 agcagaagat cacacgcttt gtgcggtata aggagatgaa tgccttcgac ggcaagacca
3241 tcgataagga gacaggcgag gtgctgcacc agaagacaca ctttcctcag ccatgggagt
3301 tctttgccca ggaagtgatg atccgggtgt ttggcaagcc tgacggcaag ccagagttcg
3361 aggaggccga taccctggag aagctgagaa cactgctggc agagaagctg agctccaggc
3421 ccgaggcagt gcacgagtac gtgaccccac tgttcgtgtc tagagccccc aacaggaaga
3481 tgagcggcca gggccacatg gagacagtga agtccgccaa gagactggac gagggcgtgt
3541 ctgtgctgag ggtgcctctg acacagctga agctgaagga tctggagaag atggtgaatc
3601 gcgagcggga gccaaagctg tatgaggccc tgaaggcaag gctggaggca cacaaggacg
3661 atcctgccaa ggcctttgcc gagccattct acaagtatga taaggccggc aacagaaccc
3721 agcaggtgaa ggccgtgagg gtggagcagg tgcagaagac aggcgtgtgg gtgcgcaacc
3781 acaatggcat cgccgacaat gctaccatgg tgcgggtgga cgtgtttgag aagggcgata
3841 agtactatct ggtgcccatc tacagctggc aggtggccaa gggcatcctg cctgatagag
3901 ccgtggtgca gggcaaggac gaggaggatt ggcagctgat cgacgattcc ttcaacttta
3961 agttctctct gcaccccaat gacctggtgg aagtgatcac caagaaggcc aggatgtttg
4021 gctacttcgc ctcctgccac cgcggcacag gcaacatcaa tatccggatc cacgacctgg
4081 atcacaagat cggcaagaac ggcatcctgg agggcatcgg cgtgaagaca gccctgagct
4141 tccagaagta tcagatcgac gagctgggca aggagatcag accttgtagg ctgaagaagc
4201 gcccacccgt gcgggaggat aagcggcccg cagcaaccaa gaaggcagga caggccaaga
4261 agaagaagta cccctatgac gtgcctgact acgccgggta tccctacgac gtgcctgatt
4321 acgccgggtc ctatccctac gacgtgccag attacgctgc agctccagca gcgaagaaaa
4381 agaagctgga ttaagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc
4441 ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga
4501 attttttgtg tctctcactc ggcggccgca ggaaccccta gtgatggagt tggccactcc
4561 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
4621 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
4681 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
4741 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
4801 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
4861 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
4921 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
4981 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
5041 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
5101 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
5161 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
5221 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
5281 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
5341 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
5401 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
5461 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
5521 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
5581 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
5641 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
5701 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
5761 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
5821 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
5881 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
5941 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
6001 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
6061 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
6121 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
6181 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
6241 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
6301 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
6361 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
6421 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
6481 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
6541 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
6601 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
6661 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
6721 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
6781 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
6841 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
6901 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
6961 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
7021 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
7081 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
7141 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
7201 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
7261 cacatgt
//
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