| 质粒名称: | pAAV-U6-sgRNA-CMV-GFP |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 85451 |
| 存储实验室: | Hetian Lei |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV |
| 载体出品公司: | Addgene |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 腺病毒表达载体AAV |
| 筛选标记: | -- |
| 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: | pCMV-EGFP |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | pCMV-EGFP |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | XbaI (未破坏) |
| 3' 克隆位点: | EcoR1(未破坏) |
| 5' 测序引物 | cgTCTAGAgGGTACCgGGGCCCgGTCGAC tagttattaa tagtaatcaa ttacgg |
| 3' 测序引物 | caGAATTCgC TGC AGg TTA TCG AGA TCT GAG TCC GGA CTT GTA |
|
文献引用方法 |
|
| 材料和方法部分: | pAAV-U6-sgRNA-CMV-GFP was a gift from Hetian Lei (Addgene plasmid # 85451 ; http://n2t.net/addgene:85451 ; RRID:Addgene_85451) |
| 参考文献部分: | The CRISPR/Cas9-created MDM2 T309G enhances vitreous-induced expression of MDM2 and proliferation and survival of cells. Duan Y, Ma G, Huang X, D'Amore PA, Zhang F, Lei H. J Biol Chem. 2016 May 31. pii: jbc.M116.729467. 10.1074/jbc.M116.729467 PubMed 27246850 |
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 pAAV-U6-sgRNA-CMV-GFP 5430 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Express sgRNA in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pAAV-U6-sgRNA-CMV-GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5430)
AUTHORS Duan Y, Ma G, Huang X, D'Amore PA, Zhang F, Lei H
TITLE The CRISPR/Cas9-created MDM2 T309G enhances vitreous-induced
expression of MDM2 and proliferation and survival of cells.
JOURNAL J Biol Chem. 2016 May 31. pii: jbc.M116.729467.
PUBMED 27246850
REFERENCE 2 (bases 1 to 5430)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5430
/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"
enhancer 592..895
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 896..1099
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 1050..1070
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
regulatory 1138..1147
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1144..1860
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(1189..1210)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(1450..1469)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 1797..1818
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 2177..2653
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(2376..2395)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
repeat_region 2693..2833
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 2693..2822
/label=AAV2 ITR
rep_origin 2908..3363
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2995..3014)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3205..3226
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(3380..3399)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 3499..3521
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(3559..3577)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 3645..3749
/gene="bla"
/label=AmpR promoter
CDS 3750..4610
/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(3968..3987)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4781..5369
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5270..5289
/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 gggtaccggg gcccggtcga ctagttatta
541 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
601 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
661 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
721 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
781 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
841 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
901 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
961 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
1021 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
1081 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc
1141 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
1201 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
1261 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
1321 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
1381 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
1441 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
1501 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
1561 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
1621 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
1681 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
1741 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
1801 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
1861 tccggactca gatctcgaga ggaggaggag gagacagaca gcaggatgcc ccacctcgac
1921 agccccggca gctcccagcc gagacgctcc ttcctctcaa gggtgatcag ggcagcgcta
1981 ccgttgcagc tgcttctgct gctgctgctg ctcctggcct gcctgctacc tgcctctgaa
2041 gatgactaca gctgcaccca ggccaacaac tttgcccgat ccttctaccc catgctgcgg
2101 tacaccaacg ggccacctcc cacctaggaa ttcgatatca agcttatcga taccgagcgc
2161 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
2221 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
2281 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
2341 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
2401 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
2461 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
2521 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
2581 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
2641 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
2701 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
2761 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
2821 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
2881 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
2941 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
3001 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
3061 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
3121 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
3181 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
3241 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
3301 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
3361 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
3421 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
3481 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
3541 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
3601 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
3661 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
3721 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
3781 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
3841 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
3901 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
3961 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
4021 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
4081 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
4141 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
4201 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
4261 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
4321 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
4381 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
4441 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
4501 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
4561 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
4621 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
4681 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
4741 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
4801 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
4861 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
4921 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
4981 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
5041 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
5101 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
5161 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
5221 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
5281 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
5341 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
5401 tggccttttg ctggcctttt gctcacatgt
//
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