LOCUS Exported 6524 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION To purify GST-tagged recombinant RIPK3 protein. ACCESSION . VERSION . KEYWORDS pGEX-4T-1-RIPK3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6524) 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 6524) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6524 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 183..211 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 219..235 /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)." CDS 258..911 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind 869..891 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 918..935 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" primer_bind complement(2574..2596) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(2741..2759) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2827..2931 /gene="bla" /label=AmpR promoter CDS 2932..3792 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(3150..3169) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3963..4551 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4452..4471 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4705..4722 /label=L4440 /note="L4440 vector, forward primer" promoter 4795..4872 /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." CDS 4873..5955 /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(4892..4911) /label=LacI-R /note="LacI, reverse primer" protein_bind 5968..5989 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6004..6034 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6042..6058 /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 6066..6082 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6066..6082 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 6078..81 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR QPSEAVVWLCRS" primer_bind complement(6098..6115) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6098..6114) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6107..6129) /label=M13/pUC Forward /note="In lacZ gene" ORIGIN 1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg 61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt 121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc 181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca 241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc 301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc 361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc 421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata 481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc 541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact 601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag 661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt 721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa 781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat 841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc 901 atcctccaaa atcggatctg gttccgcgtg gatccccgga attcccgggt cgacaaatgt 961 cgtgcgtcaa gttatggccc agcggtgccc ccgccccctt ggtgtccatc gaggaactgg 1021 agaaccagga gctcgtcggc aaaggcgggt tcggcacagt gttccgggcg caacatagga 1081 agtggggcta cgatgtggcg gtcaagatcg taaactcgaa ggcgatatcc agggaggtca 1141 aggccatggc aagtctggat aacgaattcg tgctgcgcct agaaggggtt atcgagaagg 1201 tgaactggga ccaagatccc aagccggctc tggtgactaa attcatggag aacggctcct 1261 tgtcggggct gctgcagtcc cagtgccctc ggccctggcc gctcctttgc cgcctgctga 1321 aagaagtggt gcttgggatg ttttacctgc acgaccagaa cccggtgctc ctgcaccggg 1381 acctcaagcc atccaacgtc ctgctggacc cagagctgca cgtcaagctg gcagattttg 1441 gcctgtccac atttcaggga ggctcacagt cagggacagg gtccggggag ccagggggca 1501 ccctgggcta cttggcccca gaactgtttg ttaacgtaaa ccggaaggcc tccacagcca 1561 gtgacgtcta cagcttcggg atcctaatgt gggcagtgct tgctggaaga gaagttgagt 1621 tgccaaccga accatcactc gtgtacgaag cagtgtgcaa caggcagaac cggccttcat 1681 tggctgagct gccccaagcc gggcctgaga ctcccggctt agaaggactg aaggagctaa 1741 tgcagctctg ctggagcagt gagcccaagg acagaccctc cttccaggaa tgcctaccaa 1801 aaactgatga agtcttccag atggtggaga acaatatgaa tgctgctgtc tccacggtaa 1861 aggatttcct gtctcagctc aggagcagca ataggagatt ttctatccca gagtcaggcc 1921 aaggagggac agaaatggat ggctttagga gaaccataga aaaccagcac tctcgtaatg 1981 atgtcatggt ttctgagtgg ctaaacaaac tgaatctaga ggagcctccc agctctgttc 2041 ctaaaaaatg cccgagcctt accaagagga gcagggcaca agaggagcag gttccacaag 2101 cctggacagc aggcacatct tcagattcga tggcccaacc tccccagact ccagagacct 2161 caactttcag aaaccagatg cccagcccta cctcaactgg aacaccaagt cctggacccc 2221 gagggaatca gggggctgag agacaaggca tgaactggtc ctgcaggacc ccggagccaa 2281 atccagtaac agggcgaccg ctcgttaaca tatacaactg ctctggggtg caagttggag 2341 acaacaacta cttgactatg caacagacaa ctgccttgcc cacatggggc ttggcacctt 2401 cgggcaaggg gaggggcttg cagcaccccc caccagtagg ttcgcaagaa ggccctaaag 2461 atcctgaagc ctggagcagg ccacagggtt ggtataatca tagcgggaaa taagcggccg 2521 catcgtgact gactgacgat ctgcctcgcg cgtttcggtg atgacggtga aaacctctga 2581 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 2641 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca 2701 cgtagcgata gcggagtgta taattcttga agacgaaagg gcctcgtgat acgcctattt 2761 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 2821 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 2881 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 2941 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 3001 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 3061 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 3121 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac 3181 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 3241 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 3301 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 3361 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 3421 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca 3481 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 3541 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 3601 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 3661 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 3721 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 3781 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 3841 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 3901 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 3961 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 4021 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 4081 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 4141 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 4201 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 4261 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 4321 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 4381 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 4441 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 4501 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 4561 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 4621 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 4681 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg 4741 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc 4801 atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc 4861 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct 4921 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa 4981 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg 5041 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg 5101 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg 5161 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg 5221 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg 5281 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc 5341 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca 5401 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg 5461 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg 5521 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc 5581 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc 5641 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc 5701 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg 5761 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat 5821 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc 5881 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 5941 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 6001 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 6061 tcacacagga aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc 6121 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 6181 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 6241 tgaatggcga atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc 6301 tggagtgcga tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg 6361 gttacgatgc gcccatctac accaacgtaa cctatcccat tacggtcaat ccgccgtttg 6421 ttcccacgga gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc 6481 tacaggaagg ccagacgcga attatttttg atggcgttgg aatt //