| 质粒名称: | AAV-U6-dgRNA-CAG-MPH |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 106259 |
| 存储实验室: | Juan Belmonte |
|
载体骨架基本信息 |
|
| 载体名称: | AAV |
| 完整质粒大小: | 7231bp |
| 载体类型: | 哺乳细胞、AAV、 CRISPR表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 30 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | MS2-P65-HSF1 |
| 物种来源: | H. sapiens (human), Synthetic |
| 启动子: | CAG |
| 克隆方法: | Ligation Independent Cloning |
| 5’测序引物 | 未知 |
|
文献引用方法 |
|
| 材料和方法部分: | AAV-U6-dgRNA-CAG-MPH was a gift from Juan Belmonte (Addgene plasmid # 106259 ; http://n2t.net/addgene:106259 ; RRID:Addgene_106259) |
| 参考文献部分: | In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation. Liao HK, Hatanaka F, Araoka T, Reddy P, Wu MZ, Sui Y, Yamauchi T, Sakurai M, O'Keefe DD, Nunez-Delicado E, Guillen P, Campistol JM, Wu CJ, Lu LF, Esteban CR, Izpisua Belmonte JC. Cell. 2017 Dec 14;171(7):1495-1507.e15. doi: 10.1016/j.cell.2017.10.025. Epub 2017 Dec 7. 10.1016/j.cell.2017.10.025 PubMed 29224783 |
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-U6-dgRNA-CAG-MPH 7231 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses MPH co-activator from the CAG promoter and one U6-driven
dgRNA in AAV backbone.
ACCESSION .
VERSION .
KEYWORDS AAV-U6-dgRNA-CAG-MPH
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7231)
AUTHORS Liao HK, Hatanaka F, Araoka T, Reddy P, Wu MZ, Sui Y, Yamauchi T,
Sakurai M, O'Keefe DD, Nunez-Delicado E, Guillen P, Campistol JM, Wu
CJ, Lu LF, Esteban CR, Izpisua Belmonte JC
TITLE In Vivo Target Gene Activation via CRISPR/Cas9-Mediated
Trans-epigenetic Modulation.
JOURNAL Cell. 2017 Dec 14;171(7):1495-1507.e15. doi:
10.1016/j.cell.2017.10.025. Epub 2017 Dec 7.
PUBMED 29224783
REFERENCE 2 (bases 1 to 7231)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7231
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..130
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
promoter 149..389
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 149..169
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 320..339
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 434..450
/label=MS2 stem loop
/note="stem loop that binds the bacteriophage MS2 coat
protein"
misc_RNA 504..520
/label=MS2 stem loop
/note="stem loop that binds the bacteriophage MS2 coat
protein"
enhancer 632..935
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 937..1213
/label=chicken beta-actin promoter
intron 1214..2230
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 2154..2176
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 2238..2257
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
protein_bind 2285..2309
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 2331..2717
/codon_start=1
/product="bacteriophage MS2 coat protein"
/label=MS2-N55K
/note="binds with high affinity to a specific stem-loop
structure in the viral RNA (Lim et al., 1994)"
/translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
VKAMQGLLKDGNPIPSAIAANSGIY"
CDS 2772..2792
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2808..3350
/codon_start=1
/product="C-terminal portion of the p65 subunit of mouse
NF-kappa-B"
/label=p65
/translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
FSSIADMDFSALLSQISS"
CDS 3375..3746
/codon_start=1
/product="C-terminal activation domain from the human heat
shock transcription factor HSF1"
/label=HSF1
/translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
PTISLLTGSEPPKAKDPTVS"
protein_bind complement(3780..3804)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
misc_feature 3836..4424
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3889..3909)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(4307..4318)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 4438..4486
/note="synthetic polyadenylation signal"
repeat_region 4494..4634
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4494..4623
/label=AAV2 ITR
rep_origin 4709..5164
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4796..4815)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5006..5027
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5181..5200)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5300..5322
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5360..5378)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5446..5550
/gene="bla"
/label=AmpR promoter
CDS 5551..6411
/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(5769..5788)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6582..7170
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7071..7090
/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 gaacgcgtga gggcctattt cccatgattc cttcatattt
181 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
241 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
301 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
361 cttggcttta tatatcttgt ggaaaggacg aaacaccgga agagcgagct cttctgtttc
421 agagctaggc cagcatgagg atcacccatg cctgcagggc ctagcaagtt gaaataaggc
481 tagtccgtta tcaacttggc cagcatgagg atcacccatg cctgcagggc caagtggcac
541 cgagtcggtg cttttttgca tatgctagtg cgcgttatta atagtaatca attacggggt
601 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
661 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
721 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
781 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
841 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
901 agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct
961 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat
1021 tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgcgccaggc ggggcggggc
1081 ggggcgaggg gcggggcggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
1141 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
1201 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
1261 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
1321 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
1381 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc
1441 ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg
1501 cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg
1561 gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg
1621 cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca
1681 accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct
1741 ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt
1801 gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc
1861 ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc
1921 gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag
1981 gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga
2041 aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc
2101 tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg
2161 cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt
2221 tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga
2281 attcacaagt ttgtacaaaa aagcaggctc cgaattcacc ggtcaccatg gcttcaaact
2341 ttactcagtt cgtgctcgtg gacaatggtg ggacagggga tgtgacagtg gctccttcta
2401 atttcgctaa tggggtggca gagtggatca gctccaactc acggagccag gcctacaagg
2461 tgacatgcag cgtcaggcag tctagtgccc agaagagaaa gtataccatc aaggtggagg
2521 tccccaaagt ggctacccag acagtgggcg gagtcgaact gcctgtcgcc gcttggaggt
2581 cctacctgaa catggagctc actatcccaa ttttcgctac caattctgac tgtgaactca
2641 tcgtgaaggc aatgcagggg ctcctcaaag acggtaatcc tatcccttcc gccatcgccg
2701 ctaactcagg tatctacagc gctggaggag gtggaagcgg aggaggagga agcggaggag
2761 gaggtagcgg acctaagaaa aagaggaagg tggcggccgc tggatcccct tcagggcaga
2821 tcagcaacca ggccctggct ctggccccta gctccgctcc agtgctggcc cagactatgg
2881 tgccctctag tgctatggtg cctctggccc agccacctgc tccagcccct gtgctgaccc
2941 caggaccacc ccagtcactg agcgctccag tgcccaagtc tacacaggcc ggcgagggga
3001 ctctgagtga agctctgctg cacctgcagt tcgacgctga tgaggacctg ggagctctgc
3061 tggggaacag caccgatccc ggagtgttca cagatctggc ctccgtggac aactctgagt
3121 ttcagcagct gctgaatcag ggcgtgtcca tgtctcatag tacagccgaa ccaatgctga
3181 tggagtaccc cgaagccatt acccggctgg tgaccggcag ccagcggccc cccgaccccg
3241 ctccaactcc cctgggaacc agcggcctgc ctaatgggct gtccggagat gaagacttct
3301 caagcatcgc tgatatggac tttagtgccc tgctgtcaca gatttcctct agtgggcagg
3361 gaggaggtgg aagcggcttc agcgtggaca ccagtgccct gctggacctg ttcagcccct
3421 cggtgaccgt gcccgacatg agcctgcctg accttgacag cagcctggcc agtatccaag
3481 agctcctgtc tccccaggag ccccccaggc ctcccgaggc agagaacagc agcccggatt
3541 cagggaagca gctggtgcac tacacagcgc agccgctgtt cctgctggac cccggctccg
3601 tggacaccgg gagcaacgac ctgccggtgc tgtttgagct gggagagggc tcctacttct
3661 ccgaagggga cggcttcgcc gaggacccca ccatctccct gctgacaggc tcggagcctc
3721 ccaaagccaa ggaccccact gtctccgcta gcggcatcga ttgattaatt aagaattcga
3781 cccagctttc ttgtacaaag tggtgaattc aagcttcggg gactagtcat atgataatca
3841 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
3901 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
3961 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
4021 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
4081 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
4141 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
4201 cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg
4261 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
4321 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
4381 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcacaatt ggaattcaat
4441 aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgcggc cgcaggaacc
4501 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
4561 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
4621 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
4681 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
4741 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
4801 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
4861 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
4921 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
4981 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
5041 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
5101 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
5161 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
5221 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
5281 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
5341 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
5401 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
5461 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
5521 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
5581 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
5641 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
5701 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
5761 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
5821 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
5881 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
5941 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
6001 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
6061 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
6121 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
6181 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
6241 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
6301 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
6361 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
6421 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
6481 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
6541 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
6601 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
6661 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
6721 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
6781 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
6841 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
6901 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
6961 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
7021 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
7081 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
7141 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
7201 ctggcctttt gctggccttt tgctcacatg t
//
