| 质粒名称: | Nme2Cas9_AAV |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 119924 |
| 存储实验室: | Erik Sontheimer |
|
载体骨架基本信息 |
|
| 载体名称: | AAV |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 2900 bp |
| 载体修饰: | 包含U6驱动的sgRNA盒 |
| 载体类型: | 哺乳细胞表达,AAV |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Nme2Cas9 |
| 物种来源: | N. meningitidis |
| 插入大小: | 3300 bp |
| 启动子 | U1a |
| 标签/融合蛋白: | -- |
| 克隆方法: | Gibson克隆 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | GAGGATAAGCGGCCCGCAGCAA |
| 3' 测序引物 | TTTCTTTTTCTTGGCTTGACCTGCCTTC |
|
文献引用方法 |
|
| 材料和方法部分: | Nme2Cas9_AAV was a gift from Erik Sontheimer (Addgene plasmid # 119924 ; http://n2t.net/addgene:119924 ; RRID:Addgene_119924) |
| 参考文献部分: | A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing. Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ. Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 10.1016/j.molcel.2018.12.003. 10.1016/j.molcel.2018.12.003 PubMed 30581144 |
买家导航
Nme2Cas9AAV载体基本信息
Nme2Cas9AAV载体图谱质粒图谱和多克隆位点信息
Nme2Cas9AAV载体简介
Nme2Cas9AAV载体序列
LOCUS Nme2Cas9_AAV 7270 bp ds-DNA circular SYN 18-JAN-2019
DEFINITION All-in-one AAV plasmid expressing Nme2Cas9 with sgRNA cassette.
ACCESSION .
VERSION .
KEYWORDS Nme2Cas9_AAV
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7270)
AUTHORS Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y,
Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ
TITLE A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo
Genome Editing.
JOURNAL Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi:
10.1016/j.molcel.2018.12.003.
PUBMED 30581144
REFERENCE 2 (bases 1 to 7270)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jan 18, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7270
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 1..21
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 28..75
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 3328..3375
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 3376..3465
/codon_start=1
/product="three tandem HA epitope tags"
/label=3xHA
/translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
CDS 3472..3498
/codon_start=1
/product="nuclear localization signal (Makkerh et al.,
1996)"
/label=NLS
/translation="PAAKKKKLD"
primer_bind complement(3504..3523)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_site 3569..3624
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal"
repeat_region 3637..3777
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 3637..3766
/label=AAV2 ITR
rep_origin 3852..4307
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3939..3958)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4149..4170
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4324..4343)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4443..4465
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4503..4521)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4589..4693
/gene="bla"
/label=AmpR promoter
CDS 4694..5554
/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(4912..4931)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 5725..6313
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6214..6233
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
repeat_region 6375..6504
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
misc_RNA complement(6532..6624)
/label=Nm tracrRNA
/note="trans-activating CRISPR RNA for the Neisseria
meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
promoter complement(6683..6923)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(6733..6752)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(6903..6923)
/label=hU6-F
/note="Human U6 promoter, forward primer"
regulatory 7259..7268
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
ORIGIN
1 cctaagaaga agagaaaggt ggaagataaa cgcccagcag ctacaaagaa ggcaggtcaa
61 gccaagaaaa agaaaatggc cgccttcaag cctaacccaa tcaattacat cctgggactg
121 gacatcggaa tcgcatccgt gggatgggct atggtggaga tcgacgagga ggagaatcct
181 atccggctga tcgatctggg cgtgagagtg tttgagaggg ccgaggtgcc aaagaccggc
241 gattctctgg ctatggcccg gagactggca cggagcgtga ggcgcctgac acggagaagg
301 gcacacaggc tgctgagggc acgccggctg ctgaagagag agggcgtgct gcaggcagca
361 gacttcgatg agaatggcct gatcaagagc ctgccaaaca ccccctggca gctgagagca
421 gccgccctgg acaggaagct gacaccactg gagtggtctg ccgtgctgct gcacctgatc
481 aagcaccgcg gctacctgag ccagcggaag aacgagggag agacagcaga caaggagctg
541 ggcgccctgc tgaagggagt ggccaacaat gcccacgccc tgcagaccgg cgatttcagg
601 acacctgccg agctggccct gaataagttt gagaaggagt ccggccacat cagaaaccag
661 aggggcgact atagccacac cttctcccgc aaggatctgc aggccgagct gatcctgctg
721 ttcgagaagc agaaggagtt tggcaatcca cacgtgagcg gaggcctgaa ggagggaatc
781 gagaccctgc tgatgacaca gaggcctgcc ctgtccggcg acgcagtgca gaagatgctg
841 ggacactgca ccttcgagcc tgcagagcca aaggccgcca agaacaccta cacagccgag
901 cggtttatct ggctgacaaa gctgaacaat ctgagaatcc tggagcaggg atccgagagg
961 ccactgaccg acacagagag ggccaccctg atggatgagc cttaccggaa gtctaagctg
1021 acatatgccc aggccagaaa gctgctgggc ctggaggaca ccgccttctt taagggcctg
1081 agatacggca aggataatgc cgaggcctcc acactgatgg agatgaaggc ctatcacgcc
1141 atctctcgcg ccctggagaa ggagggcctg aaggacaaga agtcccccct gaacctgagc
1201 tccgagctgc aggatgagat cggcaccgcc ttctctctgt ttaagaccga cgaggatatc
1261 acaggccgcc tgaaggacag ggtgcagcct gagatcctgg aggccctgct gaagcacatc
1321 tctttcgata agtttgtgca gatcagcctg aaggccctga gaaggatcgt gccactgatg
1381 gagcagggca agcggtacga cgaggcctgc gccgagatct acggcgatca ctatggcaag
1441 aagaacacag aggagaagat ctatctgccc cctatccctg ccgacgagat cagaaatcct
1501 gtggtgctga gggccctgtc ccaggcaaga aaagtgatca acggagtggt gcgccggtac
1561 ggatctccag cccggatcca catcgagacc gccagagaag tgggcaagag cttcaaggac
1621 cggaaggaga tcgagaagag acaggaggag aatcgcaagg atcgggagaa ggccgccgcc
1681 aagtttaggg agtacttccc taactttgtg ggcgagccaa agtctaagga catcctgaag
1741 ctgcgcctgt acgagcagca gcacggcaag tgtctgtata gcggcaagga gatcaatctg
1801 gtgcggctga acgagaaggg ctatgtggag atcgatcacg ccctgccttt ctccagaacc
1861 tgggacgatt cttttaacaa taaggtgctg gtgctgggca gcgagaacca gaataagggc
1921 aatcagacac catacgagta tttcaatggc aaggacaact ccagggagtg gcaggagttc
1981 aaggcccgcg tggagacctc tagatttccc aggagcaaga agcagcggat cctgctgcag
2041 aagttcgacg aggatggctt taaggagtgc aacctgaatg acaccagata cgtgaaccgg
2101 ttcctgtgcc agtttgtggc cgatcacatc ctgctgaccg gcaagggcaa gagaagggtg
2161 ttcgcctcta atggccagat cacaaacctg ctgaggggat tttggggact gaggaaggtg
2221 cgggcagaga atgacagaca ccacgcactg gatgcagtgg tggtggcatg cagcaccgtg
2281 gcaatgcagc agaagatcac aagattcgtg aggtataagg agatgaacgc ctttgacggc
2341 aagaccatcg ataaggagac aggcaaggtg ctgcaccaga agacccactt cccccagcct
2401 tgggagttct ttgcccagga agtgatgatc cgggtgttcg gcaagccaga cggcaagcct
2461 gagtttgagg aggccgatac cccagagaag ctgaggacac tgctggcaga gaagctgtct
2521 agcaggccag aggcagtgca cgagtacgtg accccactgt tcgtgtccag ggcacccaat
2581 cggaagatgt ctggcgccca caaggacaca ctgagaagcg ccaagaggtt tgtgaagcac
2641 aacgagaaga tctccgtgaa gagagtgtgg ctgaccgaga tcaagctggc cgatctggag
2701 aacatggtga attacaagaa cggcagggag atcgagctgt atgaggccct gaaggcaagg
2761 ctggaggcct acggaggaaa tgccaagcag gccttcgacc caaaggataa ccccttttat
2821 aagaagggag gacagctggt gaaggccgtg cgggtggaga agacccagga gagcggcgtg
2881 ctgctgaata agaagaacgc ctacacaatc gccgacaatg gcgatatggt gagagtggac
2941 gtgttctgta aggtggataa gaagggcaag aatcagtact ttatcgtgcc tatctatgcc
3001 tggcaggtgg ccgagaacat cctgccagac atcgattgca agggctacag aatcgacgat
3061 agctatacat tctgtttttc cctgcacaag tatgacctga tcgccttcca gaaggatgag
3121 aagtccaagg tggagtttgc ctactatatc aattgcgact cctctaacgg caggttctac
3181 ctggcctggc acgataaggg cagcaaggag cagcagtttc gcatctccac ccagaatctg
3241 gtgctgatcc agaagtatca ggtgaacgag ctgggcaagg agatcaggcc atgtcggctg
3301 aagaagcgcc cacccgtgcg ggaggataag cggcccgcag caaccaagaa ggcaggacag
3361 gccaagaaga agaagtaccc ctatgacgtg cctgactacg ccgggtatcc ctacgacgtg
3421 cctgattacg ccgggtccta tccctacgac gtgccagatt acgctgcagc tccagcagcg
3481 aagaaaaaga agctggatta agatcttttt ccctctgcca aaaattatgg ggacatcatg
3541 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
3601 tgttggaatt ttttgtgtct ctcactcggc ggccgcagga acccctagtg atggagttgg
3661 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
3721 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
3781 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
3841 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
3901 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
3961 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
4021 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
4081 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
4141 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
4201 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
4261 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
4321 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
4381 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
4441 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
4501 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
4561 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
4621 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
4681 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
4741 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
4801 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
4861 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
4921 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
4981 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
5041 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
5101 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
5161 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
5221 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
5281 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
5341 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
5401 tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
5461 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
5521 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
5581 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
5641 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
5701 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
5761 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
5821 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
5881 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
5941 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
6001 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
6061 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
6121 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
6181 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
6241 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
6301 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
6361 ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc
6421 gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg
6481 ccaactccat cactaggggt tcctgcggcc tctagagttt aaacaaaaaa ataaacgatg
6541 ccccttaaag cagaagcttt aaggggcaga gcgttgcggc acatcttttc agacggcctt
6601 attgtagcaa cggttctcat ttcgtttccg aaatgagaaa gggagctaca actgaagagc
6661 tggaatgctc ttccggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa
6721 tactttcaag ttacggtaag catatgatag tccattttaa aacataattt taaaactgca
6781 aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt
6841 acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa
6901 ggaatcatgg gaaataggcc ctcttcctgc ccgaccttga cgtcgactct agaatggagg
6961 cggtactatg tagatgagaa ttcaggagca aactgggaaa agcaactgct tccaaatatt
7021 tgtgattttt acagtgtagt tttggaaaaa ctcttagcct accaattctt ctaagtgttt
7081 taaaatgtgg gagccagtac acatgaagtt atagagtgtt ttaatgaggc ttaaatattt
7141 accgtaacta tgaaatgcta cgcatatcat gctgttcagg ctccgtggcc acgcaactca
7201 tacttaagca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaacaccgc
7261 caccatggtg
//
