质粒名称: | pRK5-HA-Ubiquitin-K6R |
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出品公司: | Addgene |
目录编号: | 121153 |
存储实验室: | Josef Kittler |
载体骨架基本信息 |
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载体名称: | pRK5-HA |
载体出品公司: | Ted Dawson, Addgene plasmid #17608 |
完整质粒大小: | 4996bp |
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: | Ubiquitin C / Ub |
物种来源: | H. sapiens (human) |
基因突变: | K6 mutated to an R |
克隆方法: | 限制性内切酶 |
5’克隆位点: | unknown (unknown if destroyed) |
3’克隆位点: | unknown (unknown if destroyed) |
5’测序引物 | 未知 |
文献引用方法 |
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材料和方法部分: |
pRK5-HA-Ubiquitin-K6R was a gift from Josef Kittler (Addgene plasmid # 121153 ; http://n2t.net/addgene:121153 ; RRID:Addgene_121153)
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参考文献部分: | Lysine 27 ubiquitination of the mitochondrial transport protein Miro is dependent on serine 65 of the Parkin ubiquitin ligase. Birsa N, Norkett R, Wauer T, Mevissen TE, Wu HC, Foltynie T, Bhatia K, Hirst WD, Komander D, Plun-Favreau H, Kittler JT. J Biol Chem. 2014 May 23;289(21):14569-82. doi: 10.1074/jbc.M114.563031. Epub 2014 Mar 26. 10.1074/jbc.M114.563031 PubMed 24671417 |
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LOCUS pRK5-HA-Ubiquitin-K6R 4996 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression of HA tagged ubiquitin K6R. ACCESSION . VERSION . KEYWORDS pRK5-HA-Ubiquitin-K6R SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4996) AUTHORS Birsa N, Norkett R, Wauer T, Mevissen TE, Wu HC, Foltynie T, Bhatia K, Hirst WD, Komander D, Plun-Favreau H, Kittler JT TITLE Lysine 27 ubiquitination of the mitochondrial transport protein Miro is dependent on serine 65 of the Parkin ubiquitin ligase. JOURNAL J Biol Chem. 2014 May 23;289(21):14569-82. doi: 10.1074/jbc.M114.563031. Epub 2014 Mar 26. PUBMED 24671417 REFERENCE 2 (bases 1 to 4996) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..4996 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..17 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 1..17 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" enhancer 46..425 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 426..629 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 580..600 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 626..650 /label=LNCX /note="Human CMV promoter, forward primer" promoter 860..878 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 860..877 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 891..910 /label=pMT2-F /note="Synthetic intron, forward primer" regulatory 995..1004 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1007..1033 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1064..1291 /codon_start=1 /gene="Homo sapiens UBC" /product="human ubiquitin C" /label=ubiquitin /translation="MQIFVRTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG" regulatory 1328..1337 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 1341..1475 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1378..1397) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1432..1451 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 1544..1901 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(1567..1587) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 1752..1887 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1814..1833 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(1921..1938) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1921..1937) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1930..1952) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2150..2605 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2237..2256) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2447..2468 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2622..2641) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2741..2763 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(2801..2819) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2887..2991 /gene="bla" /label=AmpR promoter CDS 2992..3852 /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(3210..3229) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4023..4611 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4512..4531 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4765..4782 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4899..4920 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4935..4965 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4973..4989 /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)." primer_bind 4978..4 /label=M13/pUC Reverse /note="In lacZ gene" ORIGIN 1 caggaaacag ctatgacatg attacgaatt aattcgagct cgcccgacat tgattattga 61 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 121 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 181 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 241 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 301 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 361 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 421 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 481 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 541 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 601 tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 661 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc 721 gggaacggtg cattggaacg cggattcccc gtgccaagag tgacgtaagt accgcctata 781 gagtctatag gcccaccccc ttggcttcgt tagaacgcgg ctacaattaa tacataacct 841 tatgtatcat acacatacga tttaggtgac actatagaat aacatccact ttgcctttct 901 ctccacaggt gtccactccc aggtccaact gcacctcggt tctatcgatt gaattcccga 961 accgacagtc ggtctcttca ccaaggccat tcgcgccacc atgggctacc cctatgatgt 1021 gcctgactac gcagatctca atggtggtgg tggtgggtcg accatgcaga tcttcgtgag 1081 gaccctgact ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt 1141 caaggcaaag atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc 1201 tgggaaacag ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac 1261 cctgcacctg gtcctccgtc tcagaggtgg ttgagcggcc gctaagtaag taaggatccc 1321 cagcttggcc gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca 1381 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 1441 ccaaactcat caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat 1501 ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc 1561 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 1621 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 1681 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 1741 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 1801 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 1861 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc 1921 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 1981 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 2041 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct 2101 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt 2161 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 2221 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 2281 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 2341 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 2401 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 2461 caaactggaa caacactcaa ccctatctcg ggctattctt ttaatttata agggattttg 2521 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 2581 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc 2641 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 2701 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 2761 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 2821 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 2881 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 2941 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 3001 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 3061 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 3121 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 3181 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 3241 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 3301 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 3361 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 3421 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 3481 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 3541 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 3601 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 3661 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 3721 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 3781 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 3841 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 3901 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 3961 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 4021 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 4081 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 4141 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 4201 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 4261 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 4321 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 4381 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 4441 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 4501 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 4561 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 4621 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 4681 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 4741 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca 4801 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 4861 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat 4921 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 4981 ggataacaat ttcaca //
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