LOCUS       sequence-394156-        6998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  double mutated phosphorylation site (kinase acitivity and MLKL 
            interaction inhibited).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6998)
  AUTHORS   Hoff J, Xiong L, Kammann T, Neugebauer S, Micheel JM, Gassler N, 
            Bauer M, Press AT
  TITLE     RIPK3 promoter hypermethylation in hepatocytes protects from bile 
            acid-induced inflammation and necroptosis.
  JOURNAL   Cell Death Dis. 2023 Apr 18;14(4):275. doi: 
            10.1038/s41419-023-05794-0.
  PUBMED    37072399
REFERENCE   2  (bases 1 to 6998)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Death 
            Dis."; date: "2023-04-18"; volume: "14(4):275. doi"; pages: " 
            10.1038/s41419-023-05794-0"
COMMENT     Sequence Label: pcDNA3-RIPK3[S199A][S227A]-FLAG
FEATURES             Location/Qualifiers
     source          1..6998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     regulatory      898..907
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             907..930
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2551..2569)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2552..2569)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2589..2606)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2595..2819
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2865..3293
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2952..2971)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3162..3183
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3302..3322)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3307..3636
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3487..3622
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3549..3568
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3703..4497
                     /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
                     LATRDIAEELGGEWANRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3757..3776)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4367..4386
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4671..4792
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4708..4727)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4762..4781
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4841..4857)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4841..4857)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4854..4876)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4865..4881
                     /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(join(4889..4895,4896..4913,4914..4919))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4934..4955
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5072..5089)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5243..5831)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5323..5342)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6002..6793,6794..6862))
                     /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     6625..6644
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6863..6967)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 gcttgccgcc
      901 accatggatt acaaggatga cgacgataag agcgcgggcg gatccgaatt ctgcagatat
      961 ccatcacact ggcggccgct cgagatgtcg tgcgtcaagt tatggcccag cggtgccccc
     1021 gcccccttgg tgtccatcga ggaactggag aaccaggagc tcgtcggcaa aggcgggttc
     1081 ggcacagtgt tccgggcgca acataggaag tggggctacg atgtggcggt caagatcgta
     1141 aactcgaagg cgatatccag ggaggtcaag gccatggcaa gtctggataa cgaattcgtg
     1201 ctgcgcctag aaggggttat cgagaaggtg aactgggacc aagatcccaa gccggctctg
     1261 gtgactaaat tcatggagaa cggctccttg tcggggctgc tgcagtccca gtgccctcgg
     1321 ccctggccgc tcctttgccg cctgctgaaa gaagtggtgc ttgggatgtt ttacctgcac
     1381 gaccagaacc cggtgctcct gcaccgggac ctcaagccat ccaacgtcct gctggaccca
     1441 gagctgcacg tcaagctggc agattttggc ctgtccacat ttcagggagg ctcacagtca
     1501 gggacagggt ccggggagcc agggggcacc ctgggctact tggccccaga actgtttgtt
     1561 aacgtaaacc ggaaggccgc cacagccagt gacgtctaca gcttcgggat cctaatgtgg
     1621 gcagtgcttg ctggaagaga agttgagttg ccaaccgaac cagccctcgt gtacgaagca
     1681 gtgtgcaaca ggcagaaccg gccttcattg gctgagctgc cccaagccgg gcctgagact
     1741 cccggcttag aaggactgaa ggagctaatg cagctctgct ggagcagtga gcccaaggac
     1801 agaccctcct tccaggaatg cctaccaaaa actgatgaag tcttccagat ggtggagaac
     1861 aatatgaatg ctgctgtctc cacggtaaag gatttcctgt ctcagctcag gagcagcaat
     1921 aggagatttt ctatcccaga gtcaggccaa ggagggacag aaatggatgg ctttaggaga
     1981 accatagaaa accagcactc tcgtaatgat gtcatggttt ctgagtggct aaacaaactg
     2041 aatctagagg agcctcccag ctctgttcct aaaaaatgcc cgagccttac caagaggagc
     2101 agggcacaag aggagcaggt tccacaagcc tggacagcag gcacatcttc agattcgatg
     2161 gcccaacctc cccagactcc agagacctca actttcagaa accagatgcc cagccctacc
     2221 tcaactggaa caccaagtcc tggaccccga gggaatcagg gggctgagag acaaggcatg
     2281 aactggtcct gcaggacccc ggagccaaat ccagtaacag ggcgaccgct cgttaacata
     2341 tacaactgct ctggggtgca agttggagac aacaactact tgactatgca acagacaact
     2401 gccttgccca catggggctt ggcaccttcg ggcaagggga ggggcttgca gcacccccca
     2461 ccagtaggtt cgcaagaagg ccctaaagat cctgaagcct ggagcaggcc acagggttgg
     2521 tataatcata gcgggaaata agggccctat tctatagtgt cacctaaatg ctagagctcg
     2581 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2641 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2701 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2761 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2821 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2881 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2941 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3001 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3061 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3121 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3181 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3241 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3301 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3361 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3421 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3481 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3541 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3601 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3661 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3721 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3781 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3841 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3901 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3961 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     4021 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     4081 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4141 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4201 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4261 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4321 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4381 gatattgctg aagagcttgg cggcgaatgg gctaaccgct tcctcgtgct ttacggtatc
     4441 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4501 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4561 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4621 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4681 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4741 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4801 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4861 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4921 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4981 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5041 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5101 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5161 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5221 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5281 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5341 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5401 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5461 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5521 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5581 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5641 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5701 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5761 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5821 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5881 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5941 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6001 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6061 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6121 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6181 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6241 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6301 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6361 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6421 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6481 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6541 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6601 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6661 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6721 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6781 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6841 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6901 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6961 ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
