LOCUS       sequence-394292-        7070 bp    DNA     circular SYN 15-APR-2025
DEFINITION  mutated ubiquitination site (ubiquitination inhibited).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7070)
  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 7070)
  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"; 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[K5A]-FLAG
FEATURES             Location/Qualifiers
     source          1..7070
                     /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        3392..3708
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3559..3694
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3621..3640
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3775..4569
                     /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(3829..3848)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4439..4458
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4743..4864
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4780..4799)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4834..4853
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4913..4929)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4913..4929)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4926..4948)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4937..4953
                     /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(4961..4967,4968..4985,4986..4991))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5006..5027
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5144..5161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5315..5903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5395..5414)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6074..6865,6866..6934))
                     /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     6697..6716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6935..7039)
                     /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 tgcgtcgcct 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 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 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 ccaggtgtgg aaagtcccca
     3481 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3541 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3601 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     3661 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     3721 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     3781 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3841 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3901 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     3961 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4021 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4081 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4141 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4201 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4261 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4321 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4381 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4441 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4501 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4561 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4621 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4681 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4741 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4801 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4861 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4921 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4981 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5041 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5101 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5161 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5221 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5281 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5341 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5401 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5461 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5521 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5581 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5641 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5701 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5761 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5821 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5881 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5941 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6001 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6061 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6121 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6181 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6241 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6301 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6361 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6421 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6481 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6541 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6601 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6661 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6721 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6781 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6841 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6901 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6961 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7021 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
