| 质粒名称: | AAV_NLS-dSaCas9-NLS-VPR |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 68495 |
| 存储实验室: | George Church |
|
载体骨架基本信息 |
|
| 载体名称: | epX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA Plasmid #61592 |
| 空载体大小: | 8613 bp |
| 完整质粒大小: | 8613 bp |
| 载体类型: | 哺乳细胞表达AAV |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 30 ℃ |
| 宿主菌: | Stbl3 |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | dSaCas9/ dSauCas9 |
| 物种来源: | Synthetic; S. aureus |
| 插入大小: | 4857 bp |
| 启动子: | CMV |
| 标签/融合蛋白: | VPR (C terminal on insert) |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | CTACTACGAAGTGAATAGCAAGTGC |
| 3’测序引物 | ACTCAGACAATGCGATGCAA |
|
文献引用方法 |
|
| 材料和方法部分: | AAV_NLS-dSaCas9-NLS-VPR was a gift from George Church (Addgene plasmid # 68495 ; http://n2t.net/addgene:68495 ; RRID:Addgene_68495) |
| 参考文献部分: | Cas9 gRNA engineering for genome editing, activation and repression. Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJ, Ebrahimkhani MR, Collins JJ, Weiss R, Church G. Nat Methods. 2015 Sep 7. doi: 10.1038/nmeth.3580. 10.1038/nmeth.3580 PubMed 26344044 |
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 AAV_NLS-dSaCas9-NLS-VPR 8602 bp ds-DNA circular SYN 05-APR-2022
DEFINITION AAV vector containing nuclease null SaCas9 fused to VPR.
ACCESSION .
VERSION .
KEYWORDS AAV_NLS-dSaCas9-NLS-VPR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8602)
AUTHORS Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian
J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJ, Ebrahimkhani MR,
Collins JJ, Weiss R, Church G
TITLE Cas9 gRNA engineering for genome editing, activation and repression.
JOURNAL Nat Methods. 2015 Sep 7. doi: 10.1038/nmeth.3580.
PUBMED 26344044
REFERENCE 2 (bases 1 to 8602)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 5, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..8602
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..141
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 12..141
/label=AAV2 ITR
enhancer 161..540
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 541..744
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 695..715
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
regulatory 763..772
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 775..795
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3976..4023
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 4054..4203
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label=VP64
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 4228..4248
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4678..5034
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/label=RelA (p65) AD
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALL"
CDS 5053..5622
/codon_start=1
/product="transcriptional activation domain from the human
herpesvirus 4 (Epstein-Barr virus) replication and
transcription activator Rta/BRLF1 (Hardwick et al., 1992;
Chavez et al., 2015)"
/label=Rta AD
/translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
DTFLNDECLLHAMHISTGLSIFDTSLF"
primer_bind complement(5650..5667)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 5656..5863
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 5876..6005
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
rep_origin complement(6067..6655)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6147..6166)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6826..7686)
/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 7449..7468
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7687..7791)
/gene="bla"
/label=AmpR promoter
primer_bind 7859..7877
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(7915..7937)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 8037..8056
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
rep_origin complement(8073..8528)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(8210..8231)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 8422..8441
/label=F1ori-R
/note="F1 origin, reverse primer"
ORIGIN
1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
121 actccatcac taggggttcc tgcggccgcc tcgaggcgtt gacattgatt attgactagt
181 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
241 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
301 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
361 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
421 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
481 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
541 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
601 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
661 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
721 tgggaggtct atataagcag agctctctgg ctaactaccg gtgccaccat ggccccaaag
781 aagaagcgga aggtcggtat ccacggagtc ccagcagcca agcggaacta catcctgggc
841 ctggccatcg gcatcaccag cgtgggctac ggcatcatcg actacgagac acgggacgtg
901 atcgatgccg gcgtgcggct gttcaaagag gccaacgtgg aaaacaacga gggcaggcgg
961 agcaagagag gcgccagaag gctgaagcgg cggaggcggc atagaatcca gagagtgaag
1021 aagctgctgt tcgactacaa cctgctgacc gaccacagcg agctgagcgg catcaacccc
1081 tacgaggcca gagtgaaggg cctgagccag aagctgagcg aggaagagtt ctctgccgcc
1141 ctgctgcacc tggccaagag aagaggcgtg cacaacgtga acgaggtgga agaggacacc
1201 ggcaacgagc tgtccaccaa agagcagatc agccggaaca gcaaggccct ggaagagaaa
1261 tacgtggccg aactgcagct ggaacggctg aagaaagacg gcgaagtgcg gggcagcatc
1321 aacagattca agaccagcga ctacgtgaaa gaagccaaac agctgctgaa ggtgcagaag
1381 gcctaccacc agctggacca gagcttcatc gacacctaca tcgacctgct ggaaacccgg
1441 cggacctact atgagggacc tggcgagggc agccccttcg gctggaagga catcaaagaa
1501 tggtacgaga tgctgatggg ccactgcacc tacttccccg aggaactgcg gagcgtgaag
1561 tacgcctaca acgccgacct gtacaacgcc ctgaacgacc tgaacaatct cgtgatcacc
1621 agggacgaga acgagaagct ggaatattac gagaagttcc agatcatcga gaacgtgttc
1681 aagcagaaga agaagcccac cctgaagcag atcgccaaag aaatcctcgt gaacgaagag
1741 gatattaagg gctacagagt gaccagcacc ggcaagcccg agttcaccaa cctgaaggtg
1801 taccacgaca tcaaggacat taccgcccgg aaagagatta ttgagaacgc cgagctgctg
1861 gatcagattg ccaagatcct gaccatctac cagagcagcg aggacatcca ggaagaactg
1921 accaatctga actccgagct gacccaggaa gagatcgagc agatctctaa tctgaagggc
1981 tataccggca cccacaacct gagcctgaag gccatcaacc tgatcctgga cgagctgtgg
2041 cacaccaacg acaaccagat cgctatcttc aaccggctga agctggtgcc caagaaggtg
2101 gacctgtccc agcagaaaga gatccccacc accctggtgg acgacttcat cctgagcccc
2161 gtcgtgaaga gaagcttcat ccagagcatc aaagtgatca acgccatcat caagaagtac
2221 ggcctgccca acgacatcat tatcgagctg gcccgcgaga agaactccaa ggacgcccag
2281 aaaatgatca acgagatgca gaagcggaac cggcagacca acgagcggat cgaggaaatc
2341 atccggacca ccggcaaaga gaacgccaag tacctgatcg agaagatcaa gctgcacgac
2401 atgcaggaag gcaagtgcct gtacagcctg gaagccatcc ctctggaaga tctgctgaac
2461 aaccccttca actatgaggt ggaccacatc atccccagaa gcgtgtcctt cgacaacagc
2521 ttcaacaaca aggtgctcgt gaagcaggaa gaagccagca agaagggcaa ccggacccca
2581 ttccagtacc tgagcagcag cgacagcaag atcagctacg aaaccttcaa gaagcacatc
2641 ctgaatctgg ccaagggcaa gggcagaatc agcaagacca agaaagagta tctgctggaa
2701 gaacgggaca tcaacaggtt ctccgtgcag aaagacttca tcaaccggaa cctggtggat
2761 accagatacg ccaccagagg cctgatgaac ctgctgcgga gctacttcag agtgaacaac
2821 ctggacgtga aagtgaagtc catcaatggc ggcttcacca gctttctgcg gcggaagtgg
2881 aagtttaaga aagagcggaa caaggggtac aagcaccacg ccgaggacgc cctgatcatt
2941 gccaacgccg atttcatctt caaagagtgg aagaaactgg acaaggccaa aaaagtgatg
3001 gaaaaccaga tgttcgagga aaagcaggcc gagagcatgc ccgagatcga aaccgagcag
3061 gagtacaaag agatcttcat caccccccac cagatcaagc acattaagga cttcaaggac
3121 tacaagtaca gccaccgggt ggacaagaag cctaatagag agctgattaa cgacaccctg
3181 tactccaccc ggaaggacga caagggcaac accctgatcg tgaacaatct gaacggcctg
3241 tacgacaagg acaatgacaa gctgaaaaag ctgatcaaca agagccccga aaagctgctg
3301 atgtaccacc acgaccccca gacctaccag aaactgaagc tgattatgga acagtacggc
3361 gacgagaaga atcccctgta caagtactac gaggaaaccg ggaactacct gaccaagtac
3421 tccaaaaagg acaacggccc cgtgatcaag aagattaagt attacggcaa caaactgaac
3481 gcccatctgg acatcaccga cgactacccc aacagcagaa acaaggtcgt gaagctgtcc
3541 ctgaagccct acagattcga cgtgtacctg gacaatggcg tgtacaagtt cgtgaccgtg
3601 aagaatctgg atgtgatcaa aaaagaaaac tactacgaag tgaatagcaa gtgctatgag
3661 gaagctaaga agctgaagaa gatcagcaac caggccgagt ttatcgcctc cttctacaac
3721 aacgatctga tcaagatcaa cggcgagctg tatagagtga tcggcgtgaa caacgacctg
3781 ctgaaccgga tcgaagtgaa catgatcgac atcacctacc gcgagtacct ggaaaacatg
3841 aacgacaaga ggccccccag gatcattaag acaatcgcct ccaagaccca gagcattaag
3901 aagtacagca cagacattct gggcaacctg tatgaagtga aatctaagaa gcaccctcag
3961 atcatcaaaa agggcaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa
4021 aagggatccg aggccagcgg ttccggacgg gctgacgcat tggacgattt tgatctggat
4081 atgctgggaa gtgacgccct cgatgatttt gaccttgaca tgcttggttc ggatgccctt
4141 gatgactttg acctcgacat gctcggcagt gacgcccttg atgatttcga cctggacatg
4201 ctgattaact ctagaagttc cggatctccg aaaaagaaac gcaaagttgg tagccagtac
4261 ctgcccgaca ccgacgaccg gcaccggatc gaggaaaagc ggaagcggac ctacgagaca
4321 ttcaagagca tcatgaagaa gtcccccttc agcggcccca ccgaccctag acctccacct
4381 agaagaatcg ccgtgcccag cagatccagc gccagcgtgc caaaacctgc cccccagcct
4441 taccccttca ccagcagcct gagcaccatc aactacgacg agttccctac catggtgttc
4501 cccagcggcc agatctctca ggcctctgct ctggctccag cccctcctca ggtgctgcct
4561 caggctcctg ctcctgcacc agctccagcc atggtgtctg cactggctca ggcaccagca
4621 cccgtgcctg tgctggctcc tggacctcca caggctgtgg ctccaccagc ccctaaacct
4681 acacaggccg gcgagggcac actgtctgaa gctctgctgc agctgcagtt cgacgacgag
4741 gatctgggag ccctgctggg aaacagcacc gatcctgccg tgttcaccga cctggccagc
4801 gtggacaaca gcgagttcca gcagctgctg aaccagggca tccctgtggc ccctcacacc
4861 accgagccca tgctgatgga ataccccgag gccatcaccc ggctcgtgac aggcgctcag
4921 aggcctcctg atccagctcc tgcccctctg ggagcaccag gcctgcctaa tggactgctg
4981 tctggcgacg aggacttcag ctctatcgcc gatatggatt tctcagcctt gctgggctct
5041 ggcagcggca gccgggattc cagggaaggg atgtttttgc cgaagcctga ggccggctcc
5101 gctattagtg acgtgtttga gggccgcgag gtgtgccagc caaaacgaat ccggccattt
5161 catcctccag gaagtccatg ggccaaccgc ccactccccg ccagcctcgc accaacacca
5221 accggtccag tacatgagcc agtcgggtca ctgaccccgg caccagtccc tcagccactg
5281 gatccagcgc ccgcagtgac tcccgaggcc agtcacctgt tggaggatcc cgatgaagag
5341 acgagccagg ctgtcaaagc ccttcgggag atggccgata ctgtgattcc ccagaaggaa
5401 gaggctgcaa tctgtggcca aatggacctt tcccatccgc ccccaagggg ccatctggat
5461 gagctgacaa ccacacttga gtccatgacc gaggatctga acctggactc acccctgacc
5521 ccggaattga acgagattct ggataccttc ctgaacgacg agtgcctctt gcatgccatg
5581 catatcagca caggactgtc catcttcgac acatctctgt tttgagaatt cctagagctc
5641 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
5701 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
5761 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
5821 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagctagag gccgcaggaa
5881 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
5941 cgaccaaagg tcgcccgacg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
6001 gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
6061 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
6121 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
6181 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
6241 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
6301 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
6361 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
6421 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
6481 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
6541 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
6601 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
6661 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
6721 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
6781 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
6841 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
6901 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
6961 accgcggctt ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
7021 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
7081 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
7141 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
7201 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
7261 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
7321 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
7381 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
7441 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
7501 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
7561 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
7621 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
7681 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
7741 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
7801 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
7861 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
7921 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
7981 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
8041 atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata ttttgttaaa
8101 attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg aaatcggcaa
8161 aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc cagtttggaa
8221 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
8281 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg
8341 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
8401 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta aggcgctggc
8461 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
8521 gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag atgcgtaagg
8581 agaaaatacc gcatcaggcg cc
//
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