LOCUS       sequence-149299-        6524 bp    DNA     circular SYN 15-APR-2025
DEFINITION  To purify GST-tagged recombinant RIPK3 4KR protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/ncb3314"; 
            journalName: "Nat Cell Biol"; date: "2016-03"; volume: "18"; issue: 
            "3"; pages: "291-302"
COMMENT     Sequence Label: pGEX-4T-1-RIPK3 4KR
FEATURES             Location/Qualifiers
     source          1..6524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..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             join(2932..3000,3001..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        join(6004..6009,6010..6027,6028..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             join(6078..6524,1..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 taaactcgcg ggcgatatcc agggaggtca
     1141 aggccatggc aagtctggat aacgaattcg tgctgcgcct agaaggggtt atcgagaagg
     1201 tgaactggga ccaagatccc cggccggctc 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 ctcgaaaatg 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 ggccctcgag
     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
//
