| 质粒名称: | AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 60229 |
| 存储实验室: | Feng Zhang |
|
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: | 2597 bp |
| 完整质粒大小: | 6394 bp |
| 载体类型: | 哺乳动物表达，小鼠靶向，AAV，Cre/Lox，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,single guide RNA,guide RNA,gRNA |
| 插入大小: | 100 bp |
| 启动子 | U6 |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | MluI(未破坏) |
| 3' 克隆位点: | XbaI (未破坏) |
| 5' 测序引物 | GAGGGCCTATTTCCCATGATTC |
|
文献引用方法 |
|
| 材料和方法部分: | AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR was a gift from Feng Zhang (Addgene plasmid # 60229 ; http://n2t.net/addgene:60229 ; RRID:Addgene_60229) |
| 参考文献部分: | CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F. Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi: 10.1016/j.cell.2014.09.014. 10.1016/j.cell.2014.09.014 PubMed 25263330 |
AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR
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AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR载体质粒基本信息
AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR质粒图谱载体图谱和AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR载体序列质粒序列多克隆位点信息
-pCBh-Cre-WPRE-hGHpA-ITR质粒图谱.png)
AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR质粒载体简介
AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR质粒序列载体序列
LOCUS AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR 6362 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses Cre recombinase from the Cbh promoter and one U6-driven
sgRNA. AAV backbone with SapI spacer for sgRNA cloning..
ACCESSION .
VERSION .
KEYWORDS AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6362)
AUTHORS Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE,
Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S,
Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A,
Feng G, Sharp PA, Zhang F
TITLE CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.
JOURNAL Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi:
10.1016/j.cell.2014.09.014.
PUBMED 25263330
REFERENCE 2 (bases 1 to 6362)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6362
/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 145..385
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 145..165
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 316..335
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 412..487
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 577..862
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 864..1139
/label=chicken beta-actin promoter
intron 1140..1367
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 1388..1408
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2435..2461
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
misc_feature 2489..3077
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(2542..2562)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(2960..2971)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 3109..3585
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(3308..3327)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
repeat_region 3625..3765
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 3625..3754
/label=AAV2 ITR
rep_origin 3840..4295
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3927..3946)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4137..4158
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4312..4331)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4431..4453
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4491..4509)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4577..4681
/gene="bla"
/label=AmpR promoter
CDS 4682..5542
/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(4900..4919)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 5713..6301
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6202..6221
/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 gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagag
421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
481 tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgtagcg
541 cgtgcgccaa ttctgcagac aaattctaga ggtacccgtt acataactta cggtaaatgg
601 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg
661 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
721 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
781 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
841 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
901 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
961 gatgggggcg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg
1021 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
1081 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
1141 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
1201 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
1261 cgggctgtaa ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat
1321 taatgtttaa ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc
1381 caccatgccc aagaagaaga ggaaggtgtc caatctcctg actgttcacc agaacctccc
1441 tgcgctgcca gtagatgcca ctagcgatga ggtcaggaaa aatctcatgg atatgtttag
1501 ggatagacag gcgttttctg aacacacctg gaaaatgctg cttagcgtgt gccgatcctg
1561 ggcagcctgg tgtaagctga acaatcgcaa atggttcccc gccgagccgg aggacgtgcg
1621 cgattacctg ctgtatctcc aggcaagagg gctggctgtc aagactatcc agcagcactt
1681 gggccaactg aatatgctgc atcgacgcag cgggctcccc cggcctagcg attcaaacgc
1741 agtctccctt gttatgagga gaattagaaa ggaaaacgta gatgcgggtg agagggctaa
1801 gcaggctctc gcttttgagc ggactgattt cgaccaggtc agatccctga tggagaacag
1861 cgatcggtgc caggacatca ggaacctcgc atttctggga attgcatata acacacttct
1921 gcgcatagct gagatcgccc ggatcagagt gaaagacatc agtcgaacgg acggcggccg
1981 gatgcttatt catattggac gcacaaagac attggtcagc accgctggcg ttgaaaaggc
2041 cttgtccctg ggcgtaacga agctggtgga aagatggatc tcagtgtccg gcgtggctga
2101 cgaccctaat aattacttgt tctgtcgagt gagaaaaaac ggagtcgccg cgccctctgc
2161 caccagccaa ttgagtacac gggcccttga agggatcttt gaggcaaccc accgactcat
2221 atacggagcc aaggatgaca gtggccagag gtatctcgcc tggtcaggtc attctgctag
2281 ggtgggggcc gcacgagaca tggcgcgggc aggagtctcc ataccagaga ttatgcaagc
2341 tggaggttgg acaaatgtga acatcgttat gaactatatc cgcaatcttg actctgaaac
2401 cggggccatg gtgagactgc tcgaagatgg tgactaccca tacgatgttc cagattacgc
2461 ttaagaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
2521 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
2581 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
2641 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
2701 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
2761 ttccggaact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
2821 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
2881 gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac
2941 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
3001 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
3061 ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc
3121 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
3181 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
3241 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
3301 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
3361 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
3421 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
3481 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
3541 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta
3601 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
3661 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
3721 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
3781 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
3841 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
3901 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
3961 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
4021 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
4081 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
4141 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
4201 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
4261 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
4321 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
4381 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
4441 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
4501 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
4561 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
4621 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
4681 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
4741 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
4801 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
4861 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
4921 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
4981 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
5041 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
5101 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
5161 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
5221 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
5281 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
5341 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
5401 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
5461 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
5521 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
5581 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
5641 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
5701 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
5761 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
5821 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
5881 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
5941 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
6001 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
6061 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
6121 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
6181 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
6241 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
6301 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
6361 gt
//
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