| 质粒名称: | AAV:ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 60231 |
| 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: | 6562 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: | 102 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 | ctggtcctaaaacccacttgc |
|
文献引用方法 |
|
| 材料和方法部分: | AAV:ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR was a gift from Feng Zhang (Addgene plasmid # 60231 ; http://n2t.net/addgene:60231 ; RRID:Addgene_60231) |
| 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)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortP
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
-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR质粒图谱.png)
载体简介
载体序列
LOCUS AAV:ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR 6562 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses Cre recombinase and KASH-tagged EGFP from the hSyn
promoter and one U6-driven sgRNA. AAV backbone with SapI spacer for
sgRNA cloning..
ACCESSION .
VERSION .
KEYWORDS AAV:ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6562)
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 6562)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6562
/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
promoter 156..396
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 156..176
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 327..346
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 423..498
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 532..979
/label=hSyn promoter
/note="human synapsin I promoter; confers neuron-specific
expression (Kugler et al., 2003)"
CDS 1002..1022
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2049..2075
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 2091..2144
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 2145..2858
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(2187..2208)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(2448..2467)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 2795..2816
/label=EGFP-C
/note="EGFP, forward primer"
misc_feature 3150..3738
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3203..3223)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(3621..3632)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
repeat_region 3825..3965
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 3825..3954
/label=AAV2 ITR
rep_origin 4040..4495
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4127..4146)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4337..4358
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4512..4531)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4631..4653
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4691..4709)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4777..4881
/gene="bla"
/label=AmpR promoter
CDS 4882..5742
/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(5100..5119)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 5913..6501
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6402..6421
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt
181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggaaga gcgagctctt
421 ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
481 gtggcaccga gtcggtgctt tttttctaga ctgcagaggg ccctgcgtat gagtgcaagt
541 gggttttagg accaggatga ggcggggtgg gggtgcctac ctgacgaccg accccgaccc
601 actggacaag cacccaaccc ccattcccca aattgcgcat cccctatcag agagggggag
661 gggaaacagg atgcggcgag gcgcgtgcgc actgccagct tcagcaccgc ggacagtgcc
721 ttcgcccccg cctggcggcg cgcgccaccg ccgcctcagc actgaaggcg cgctgacgtc
781 actcgccggt cccccgcaaa ctccccttcc cggccacctt ggtcgcgtcc gcgccgccgc
841 cggcccagcc ggaccgcacc acgcgaggcg cgagataggg gggcacgggc gcgaccatct
901 gcgctgcggc gccggcgact cagcgctgcc tcagtctgcg gtgggcagcg gaggagtcgt
961 gtcgtgcctg agagcgcagt cgagaaggta ccgccaccat gcccaagaag aagaggaagg
1021 tgtccaatct cctgactgtt caccagaacc tccctgcgct gccagtagat gccactagcg
1081 atgaggtcag gaaaaatctc atggatatgt ttagggatag acaggcgttt tctgaacaca
1141 cctggaaaat gctgcttagc gtgtgccgat cctgggcagc ctggtgtaag ctgaacaatc
1201 gcaaatggtt ccccgccgag ccggaggacg tgcgcgatta cctgctgtat ctccaggcaa
1261 gagggctggc tgtcaagact atccagcagc acttgggcca actgaatatg ctgcatcgac
1321 gcagcgggct cccccggcct agcgattcaa acgcagtctc ccttgttatg aggagaatta
1381 gaaaggaaaa cgtagatgcg ggtgagaggg ctaagcaggc tctcgctttt gagcggactg
1441 atttcgacca ggtcagatcc ctgatggaga acagcgatcg gtgccaggac atcaggaacc
1501 tcgcatttct gggaattgca tataacacac ttctgcgcat agctgagatc gcccggatca
1561 gagtgaaaga catcagtcga acggacggcg gccggatgct tattcatatt ggacgcacaa
1621 agacattggt cagcaccgct ggcgttgaaa aggccttgtc cctgggcgta acgaagctgg
1681 tggaaagatg gatctcagtg tccggcgtgg ctgacgaccc taataattac ttgttctgtc
1741 gagtgagaaa aaacggagtc gccgcgccct ctgccaccag ccaattgagt acacgggccc
1801 ttgaagggat ctttgaggca acccaccgac tcatatacgg agccaaggat gacagtggcc
1861 agaggtatct cgcctggtca ggtcattctg ctagggtggg ggccgcacga gacatggcgc
1921 gggcaggagt ctccatacca gagattatgc aagctggagg ttggacaaat gtgaacatcg
1981 ttatgaacta tatccgcaat cttgactctg aaaccggggc catggtgaga ctgctcgaag
2041 atggtgacta cccatacgat gttccagatt acgctgctag cggcagtgga gagggcagag
2101 gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagtgagc aagggcgagg
2161 agctgttcac cggggtggta cccatcctgg tcgagctgga cggcgacgta aacggccaca
2221 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
2281 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
2341 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
2401 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
2461 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
2521 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
2581 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
2641 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
2701 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
2761 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
2821 ccgccgggat cactctcggc atggacgagc tgtacaagtc cggactcaga tctcgagagg
2881 aggaggagga gacagacagc aggatgcccc acctcgacag ccccggcagc tcccagccga
2941 gacgctcctt cctctcaagg gtgatcaggg cagcgctacc gttgcagctg cttctgctgc
3001 tgctgctgct cctggcctgc ctgctacctg cctctgaaga tgactacagc tgcacccagg
3061 ccaacaactt tgcccgatcc ttctacccca tgctgcggta caccaacggg ccacctccca
3121 cctaggaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
3181 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
3241 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
3301 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
3361 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
3421 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
3481 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
3541 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
3601 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
3661 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
3721 tttgggccgc ctccccgcat cgataccgag cgctaataaa agatctttat tttcattaga
3781 tctgtgtgtt ggttttttgt gtcacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
3841 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
3901 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
3961 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
4021 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
4081 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
4141 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
4201 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
4261 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
4321 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
4381 ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
4441 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
4501 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
4561 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
4621 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
4681 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
4741 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
4801 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
4861 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
4921 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
4981 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
5041 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
5101 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
5161 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
5221 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
5281 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
5341 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
5401 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
5461 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
5521 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
5581 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
5641 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
5701 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
5761 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
5821 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
5881 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
5941 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
6001 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
6061 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
6121 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
6181 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
6241 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
6301 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
6361 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
6421 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
6481 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
6541 tgctggcctt ttgctcacat gt
//
