- 商品货号:FH1428
- 规格:10μl质粒
- 启动子:CMV promoter
- 复制子:pUC Ori,F1 Ori,SV40 Ori
- 终止子:HSV TK poly(A) signal
- 质粒分类:哺乳细胞质粒;哺乳编辑质粒;干扰RNA质粒
- 质粒大小:5759bp
- 原核抗性:壮观霉素Spe
- 筛选标记:杀稻瘟菌素Blast
- 克隆菌株:DH5α等大肠杆菌
- 表达宿主:293T等哺乳细胞
- 诱导方式:无需诱导
- 5测序引物:pEGFP-C-5
- 3测序引物:HSVtk-pA-R:GTCTCCTTCCGTGTTTCAG
- 备注:可连入miRNA片段
- 质粒简介: pCDNA6.2-GW-EmGFP-miR-Control质粒是在pcDNA6.2-GW/EmGFP-miR线性化载体的基础上连入了一个小片段而成。如果需要连入其它miRNA片段,可通过引物退火加酶切连接的方式连入。 转染后可立即表达 miRNA。 所表达的 miRNA 在核内经内源细胞机制(包括 Drosha 酶)加工后,转运至细胞质,由 Dicer 酶进一步加工)。 然后,完成加工的 miRNA 结合到 RISC,在此处其功能类似于 siRNA,使 mRNA 靶标被切割。
带有 EmGFP 的 BLOCK-iT™ Pol II miR RNAi 表达载体试剂盒将传统 RNAi 载体的优点(表达稳定并能够使用病毒导入)以及组织特异性表达和同一转录本多靶点抑制的功能集于一身。 带有 EmGFP 的 BLOCK-iT™ Pol II miR RNAi 表达载体试剂盒含有的 pcDNA6.2-GW/EmGFP-miR 载体设计用于表达人工 miRNA,该 miRNA 经工程化改造,与目的基因序列有 100% 的同源性并能引起靶标分子的切割。 该载体包括内源性 miRNA 的侧翼序列和环序列,内源性 miRNA 可指导从较长的 Pol II 转录本 (pre-miRNA) 上切除工程化的 miRNA。 使用 一流的 BLOCK-iT RNAi Designer,超过 70% 的重组体能产生超过 70% 的抑制效率。 工程化 miRNA 的 Pol II 表达可实现:
在 CMV 即刻早期启动子作用下高效表达,可通过 MultiSite Gateway重组选择组织特异性启动子和其它受调控的启动子;
在 pcDNA™6.2-GW/EmGFP-miR 载体中同-顺反子表达 Emerald GFP (EmGFP) 报告基因,使 EmGFP 的表达与目的 miRNA 抑制活性之间具有极强的相关性;
与用于基因表达的多种 Invitrogen Gateway® 目的 (DEST) 载体兼容;包括用于稳定转导分裂细胞、非分裂细胞和原代细胞类型的慢病毒载体,用于染色体单位点整合的 Flp-In™ 系统,以及替代报告基因融合构建体;
可在同一转录本表达的工程化 miRNA 多于一种,从而可同时抑制多个基因并产生合成表型;- 质粒图谱1: png/1-1F91Z92401607.png
质粒序列: 点击查看序列LOCUS Exported 5759 bp ds-DNA circular SYN 19-SEP-2017DEFINITION synthetic circular DNAKEYWORDS pCDNA6.2-GW-EmGFP-miR-ControlSOURCE synthetic DNA constructORGANISM synthetic DNA constructREFERENCE 1 (bases 1 to 5759)TITLE Direct SubmissionJOURNAL Exported Sep 19, 2017 from MiaoLingPlasmidhttp://www.fenghbio.cnFEATURES Location/Qualifierssource 1..5759/organism="synthetic DNA construct"/mol_type="other DNA"enhancer 4..383/label=CMV enhancer/note="human cytomegalovirus immediate early enhancer"promoter 384..587/label=CMV promoter/note="human cytomegalovirus (CMV) immediate earlypromoter"promoter 632..650/label=T7 promoter/note="promoter for bacteriophage T7 RNA polymerase"protein_bind 680..704/gene="mutant version of attB"/label=attB1/bound_moiety="BP Clonase(TM)"/note="recombination site for the Gateway(R) BP reaction"CDS 713..1432/codon_start=1/product="Emerald GFP"/label=EmGFP/note="mammalian codon-optimized"/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"ncRNA 1492..1518/label=5' miR-155/note="sequence upstream of the precursor of mouse miR-155microRNA (Uva et al., 2013)"/ncRNA_class="miRNA"misc_feature 1519..1581/label=ControlncRNA 1582..1623/label=3' miR-155/note="sequence downstream of the precursor of mousemiR-155 microRNA (Uva et al., 2013)"/ncRNA_class="miRNA"protein_bind complement(1652..1676)/gene="mutant version of attB"/label=attB2/bound_moiety="BP Clonase(TM)"/note="recombination site for the Gateway(R) BP reaction"polyA_signal 1705..1976/label=HSV TK poly(A) signal/note="herpes simplex virus thymidine kinasepolyadenylation signal (Cole and Stacy, 1985)"rep_origin 2088..2516/direction=RIGHT/label=f1 ori/note="f1 bacteriophage origin of replication; arrowindicates direction of (+) strand synthesis"promoter 2530..2859/label=SV40 promoter/note="SV40 enhancer and early promoter"rep_origin 2710..2845/label=SV40 ori/note="SV40 origin of replication"promoter 2907..2954/label=EM7 promoter/note="synthetic bacterial promoter "CDS 2973..3371/codon_start=1/gene="Aspergillus terreus BSD"/product="blasticidin S deaminase"/label=Blast/note="confers resistance to blasticidin"/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGVNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIKAIVKDSDGQPTAVGIRELLPSGYVWEG"polyA_signal 3529..3659/label=SV40 poly(A) signal/note="SV40 polyadenylation signal"rep_origin complement(3856..4444)/direction=LEFT/label=ori/note="high-copy-number ColE1/pMB1/pBR322/pUC origin ofreplication"CDS complement(4540..5331)/codon_start=1/gene="aadA"/product="aminoglycoside adenylyltransferase (Murphy,1985)"/label=Spe/note="confers resistance to spectinomycin andstreptomycin"/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"ORIGIN1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta601 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagtcccaag661 ctggctagtt aagctatcaa caagtttgta caaaaaagca ggctttaaaa ccatggtgag721 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt781 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct841 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac901 caccttcacc tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga961 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga1021 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg1081 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga1141 gtacaactac aacagccaca aggtctatat caccgccgac aagcagaaga acggcatcaa1201 ggtgaacttc aagacccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta1261 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag1321 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga1381 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aagctaagca1441 cttcgtggcc gtcgatcgtt taaagggagg tagtgagtcg accagtggat cctggaggct1501 tgctgaaggc tgtatgctgt gtcagtttgt caaatacccc gttttggcca ctgactgacg1561 gggtattaca aactgacaca ggacacaagg cctgttacta gcactcacat ggaacaaatg1621 gcccagatct ggccgcactc gagatatcta gacccagctt tcttgtacaa agtggttgat1681 ctagagggcc cgcggttcgc tgatggggga ggctaactga aacacggaag gagacaatac1741 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac gggtgttggg1801 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg1861 tgaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt1921 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcatcc1981 cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc2041 aaaatctctg atggatctgc gcagctgggg ctctaggggg tatccccacg cgccctgtag2101 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag2161 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt2221 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca2281 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata2341 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca2401 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc2461 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt2521 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt2581 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca2641 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta2701 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga2761 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag2821 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata2881 tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca2941 tagtataata cgacaaggtg aggaactaaa ccatggccaa gcctttgtct caagaagaat3001 ccaccctcat tgaaagagca acggctaca
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