LOCUS       sequence-149277-        6999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To overexpress RIPK3 K50A in Mammalian Cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6999)
  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 6999)
  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"; doi: "10.1038/ncb3314"; 
            journalName: "Nat Cell Biol"; date: "2016-03"; volume: "18"; issue: 
            "3"; pages: "291-302"
COMMENT     Sequence Label: pcDNA3-FLAG-RIPK3 K50A
FEATURES             Location/Qualifiers
     source          1..6999
                     /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..3637
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3488..3623
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3550..3569
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3704..4498
                     /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(3758..3777)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4368..4387
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4672..4793
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4709..4728)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4763..4782
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4842..4858)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4842..4858)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4855..4877)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4866..4882
                     /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(4890..4896,4897..4914,4915..4920))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4935..4956
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5073..5090)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5244..5832)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5324..5343)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6003..6794,6795..6863))
                     /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     6626..6645
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6864..6968)
                     /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 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 cgcgatcgta
     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 ggaaggcctc cacagccagt gacgtctaca gcttcgggat cctaatgtgg
     1621 gcagtgcttg ctggaagaga agttgagttg ccaaccgaac catcactcgt 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 gcatcccttt agggttccga tttagtgctt tacggcacct
     3061 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3121 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3181 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttggggat
     3241 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3301 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccaggcag gcagaagtat
     3361 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3421 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3481 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3541 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     3601 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     3661 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     3721 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3781 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3841 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     3901 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3961 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     4021 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     4081 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     4141 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     4201 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     4261 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     4321 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     4381 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     4441 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     4501 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4561 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4621 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4681 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4741 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4801 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4861 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4921 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4981 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5041 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5101 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5161 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5221 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5281 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5341 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5401 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
     5461 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5521 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5581 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5641 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5701 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5761 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5821 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5881 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5941 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6001 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6061 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6121 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6181 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6241 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6301 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6361 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6421 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6481 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6541 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6601 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6661 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6721 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6781 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6841 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6901 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6961 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
