LOCUS       sequence-150436-        7417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To overexpress RIPK1 K316R in Mammalian Cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7417)
  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 7417)
  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-RIPK1 K316R
FEATURES             Location/Qualifiers
     source          1..7417
                     /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"
     primer_bind     complement(3011..3028)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3017..3241
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3287..3715
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3374..3393)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3584..3605
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3724..3744)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3729..4058
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3909..4044
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3971..3990
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4125..4919
                     /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(4179..4198)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4789..4808
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5093..5214
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5130..5149)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5184..5203
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5263..5279)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5263..5279)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5276..5298)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5287..5303
                     /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(5311..5317,5318..5335,5336..5341))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5356..5377
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5494..5511)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5665..6250)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5745..5764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6421..7212,7213..7281))
                     /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     7044..7063
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7282..7386)
                     /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 catgcaacca
      961 gacatgtcct tgaatgtcat taagatgaaa tccagtgact tcctggagag tgcagaactg
     1021 gacagcggag gctttgggaa ggtgtctctg tgtttccaca gaacccaggg actcatgatc
     1081 atgaaaacag tgtacaaggg gcccaactgc attgagcaca acgaggccct cttggaggag
     1141 gcgaagatga tgaacagact gagacacagc cgggtggtga agctcctggg cgtcatcata
     1201 gaggaaggga agtactccct ggtgatggag tacatggaga agggcaacct gatgcacgtg
     1261 ctgaaagccg agatgagtac tccgctttct gtaaaaggaa ggataatttt ggaaatcatt
     1321 gaaggaatgt gctacttaca tggaaaaggc gtgatacaca aggacctgaa gcctgaaaat
     1381 atccttgttg ataatgactt ccacattaag atcgcagacc tcggccttgc ctcctttaag
     1441 atgtggagca aactgaataa tgaagagcac aatgagctga gggaagtgga cggcaccgct
     1501 aagaagaatg gcggcaccct ctactacatg gcgcccgagc acctgaatga cgtcaacgca
     1561 aagcccacag agaagtcgga tgtgtacagc tttgctgtag tactctgggc gatatttgca
     1621 aataaggagc catatgaaaa tgctatctgt gagcagcagt tgataatgtg cataaaatct
     1681 gggaacaggc cagatgtgga tgacatcact gagtactgcc caagagaaat tatcagtctc
     1741 atgaagctct gctgggaagc gaatccggaa gctcggccga catttcctgg cattgaagaa
     1801 aaatttaggc ctttttattt aagtcaatta gaagaaagtg tagaagagga cgtgaagagt
     1861 ttaaagaaag agtattcaaa cgaaaatgca gttgtgagga gaatgcagtc tcttcaactt
     1921 gattgtgtgg cagtaccttc aagccggtca aattcagcca cagaacagcc tggttcactg
     1981 cacagttccc agggacttgg gatgggtcct gtggaggagt cctggtttgc tccttccctg
     2041 gagcacccac aagaagagaa tgagcccagc ctgcagagta aactccaaga cgaagccaac
     2101 taccatcttt atggcagccg catggacagg cagacgaaac agcagcccag acagaatgtg
     2161 gcttacaaca gagaggagga aaggagacgc agggtctccc atgacccttt tgcacagcaa
     2221 agaccttacg agaattttca gaatacagag ggaaaaggca ctgcttattc cagtgcagcc
     2281 agtcatggta atgcagtgca ccagccctca gggctcacca gccaacctca agtactgtat
     2341 cagaacaatg gattatatag ctcacatggc tttggaacaa gaccactgga tccaggaaca
     2401 gcaggtccca gagtttggta caggccaatt ccaagtcata tgcctagtct gcataatatc
     2461 ccagtgcctg agaccaacta tctaggaaat acacccacca tgccattcag ctccttgcca
     2521 ccaacagatg aatctataaa atataccata tacaatagta ctggcattca gattggagcc
     2581 tacaattata tggagattgg tgggacgagt tcatcactac tagacagcac aaatacgaac
     2641 ttcaaagaag agccagctgc taagtaccaa gctatctttg ataataccac tagtctgacg
     2701 gataaacacc tggacccaat cagggaaaat ctgggaaagc actggaaaaa ctgtgcccgt
     2761 aaactgggct tcacacagtc tcagattgat gaaattgacc atgactatga gcgagatgga
     2821 ctgaaagaaa aggtttacca gatgctccaa aagtgggtga tgagggaagg cataaaggga
     2881 gccacggtgg ggaagctggc ccaggcgctc caccagtgtt ccaggatcga ccttctgagc
     2941 agcttgattt acgtcagcca gaactaactc gagcatgcat ctagagggcc cgtttaaacc
     3001 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3061 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3121 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3181 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3241 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3301 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3361 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3421 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3481 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3541 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3601 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3661 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3721 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3781 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3841 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3901 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3961 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     4021 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     4081 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4141 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4201 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4261 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4321 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4381 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4441 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4501 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4561 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4621 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4681 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4741 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4801 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4861 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4921 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4981 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5041 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     5101 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5161 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5221 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5281 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5341 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5401 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5461 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5521 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5581 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5641 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5701 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5761 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5821 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5881 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5941 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6001 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6061 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6121 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6181 acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6241 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6301 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6361 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6421 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6481 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6541 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6601 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6661 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6721 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6781 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6841 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6901 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6961 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7021 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7081 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7141 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7201 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7261 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7321 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7381 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
