质粒名称: | pcDNA3-HA-mRipk3 |
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出品公司: | addgene |
目录编号: | 78805 |
存储实验室: | Jaewhan Song |
载体骨架基本信息 |
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载体名称: | pcDNA3-HA |
完整质粒大小: | 6885 bp |
载体类型: | 哺乳细胞表达 |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | RIPK3 |
物种来源: | M. musculus (mouse) |
基因突变: | Ripk3 (a.k.a. 2610528K09Rik, Rip3) |
启动子: | CMV |
克隆方法: | 限制性内切酶 |
5’克隆位点: | 未知(未破坏) |
3’克隆位点: | 未知 (未破坏) |
5’测序引物 | CMV-F |
3’测序引物 | BGHrev |
文献引用方法 |
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材料和方法部分: | pcDNA3-HA-mRipk3 was a gift from Jaewhan Song (Addgene plasmid # 78805 ; http://n2t.net/addgene:78805 ; RRID:Addgene_78805) |
参考文献部分: | CHIP controls necroptosis through ubiquitylation- and lysosome-dependent degradation of RIPK3. Seo J, Lee EW, Sung H, Seong D, Dondelinger Y, Shin J, Jeong M, Lee HK, Kim JH, Han SY, Lee C, Seong JK, Vandenabeele P, Song J. Nat Cell Biol. 2016 Mar;18(3):291-302. doi: 10.1038/ncb3314. Epub 2016 Feb 22. 10.1038/ncb3314 PubMed 26900751 |
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载体基本信息
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载体序列
LOCUS pcDNA3-HA-mRipk3 6885 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION To overexpress mRipk3 in Mammalian Cells. ACCESSION . VERSION . KEYWORDS pcDNA3-HA-mRipk3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6885) AUTHORS Seo J, Lee EW, Sung H, Seong D, Dondelinger Y, Shin J, Jeong M, Lee HK, Kim JH, Han SY, Lee C, Seong JK, Vandenabeele P, Song J TITLE CHIP controls necroptosis through ubiquitylation- and lysosome-dependent degradation of RIPK3. JOURNAL Nat Cell Biol. 2016 Mar;18(3):291-302. doi: 10.1038/ncb3314. Epub 2016 Feb 22. PUBMED 26900751 REFERENCE 2 (bases 1 to 6885) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..6885 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 910..936 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 2298..2309 /codon_start=1 /product="WELQut protease recognition and cleavage site" /label=WELQut site /translation="WELQ" promoter complement(2437..2455) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2438..2455) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2475..2492) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2481..2705 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2751..3179 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2838..2857) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3048..3069 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3188..3208) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3193..3523 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3374..3509 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3436..3455 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3590..4384 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3644..3663) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4254..4273 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4558..4679 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4595..4614) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4649..4668 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4728..4744) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4728..4744) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4741..4763) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4752..4768 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4776..4806) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4821..4842 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4959..4976) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5130..5718) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5210..5229) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5889..6749) /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 6512..6531 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6750..6854) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 901 atggcctcct acccttatga tgtgccagat tatgcctctc ccgaattcat gtcttctgtc 961 aagttatggc ctactggtgc gtcagcggtt cctctggtga gccgtgaaga actgaagaag 1021 ctggagtttg tgggcaaagg agggttcgga gtcgtgttcc gggcacacca cagaacatgg 1081 aaccatgatg tagcagtcaa gatcgtgaac tcgaagaaga tatcctggga ggtgaaggct 1141 atggttaatc ttcgtaatga gaacgttctg ctcctgctgg gggtcactga ggacctccag 1201 tgggacttcg tgtccgggca ggctctggtg acaagattca tggagaatgg ctccctcgca 1261 gggctgctgc aacccgagtg ccctcggccc tggccactcc tctgtcgcct gctgcaggaa 1321 gtggtgctgg ggatgtgcta cctacacagc ttgaaccctc cgctcctgca ccgggacctc 1381 aagccctcta acattctgct ggatccagag ctccacgcca agctagcaga ttttggcctg 1441 tccacgtttc agggagggtc ccagtcaggg tcaggatcag gatcaggatc cagggactct 1501 gggggcaccc tagcgtactt ggacccagag ctgttattta atgtcaacct gaaggcttct 1561 aaagcgagtg atgtctacag ctttgggatc ctcgtgtggg cagtgctggc tggcagagaa 1621 gctgagttgg tagacaagac ttcactaatc cgggaaacag tgtgtgacag gcagagtcgt 1681 cctccactga cagagctgcc tccaggtagc cctgagactc ctggcttgga aaaactgaag 1741 gagttaatga ttcattgctg gggttcccag tccgaaaaca ggccatcctt ccaggactgc 1801 gaaccaaaaa ccaatgaagt ttacaatctg gtaaaggaca aggtagatgc tgctgtctcc 1861 gaggtaaagc attatctgtc tcagcacaga agcagcggca gaaacttgtc tgccagagag 1921 ccaagccaaa gaggcacaga aatggattgc ccgagggaaa ccatggtttc taaaatgctg 1981 gaccgcctgc atttggagga accctccgga ccagttcctg gaaaatgtcc tgagaggcaa 2041 gcacaggaca catcagttgg gcctgccaca ccagcaagga catcttctga ccccgtggct 2101 ggcactcctc agattccaca tactttaccc ttcagaggca caacacctgg gccagtcttt 2161 actgagactc ccggtcctca cccccaaagg aatcagggag atggaagaca cggcactcct 2221 tggtatccct ggaccccacc gaatccaatg acagggccac cggctctcgt cttcaacaac 2281 tgttctgaag tgcagattgg gaactacaac tccttggtag caccaccaag aactactgcc 2341 tcaagttcgg ccaagtatga ccaagcacag ttcggcaggg gtaggggctg gcagcccttc 2401 cacaagtagc tcgagcatgc atctagaggg ccctattcta tagtgtcacc taaatgctag 2461 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 2521 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 2581 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 2641 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 2701 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg 2761 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 2821 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 2881 ctttccccgt caagctctaa atcggggcat ccctttaggg ttccgattta gtgctttacg 2941 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg 3001 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 3061 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt 3121 ggggatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta 3181 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc aggcaggcag 3241 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc 3301 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc 3361 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg 3421 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca 3481 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg 3541 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca 3601 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg 3661 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg 3721 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc 3781 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt 3841 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc 3901 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca 3961 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc 4021 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg 4081 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct 4141 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc 4201 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc 4261 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta 4321 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt 4381 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga 4441 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac 4501 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac 4561 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 4621 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 4681 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt 4741 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 4801 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 4861 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 4921 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 4981 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 5041 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 5101 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 5161 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 5221 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 5281 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg 5341 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 5401 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 5461 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 5521 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 5581 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 5641 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 5701 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 5761 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 5821 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5881 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5941 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 6001 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 6061 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 6121 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 6181 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 6241 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 6301 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 6361 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 6421 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 6481 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 6541 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 6601 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 6661 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 6721 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 6781 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 6841 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc //
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