LOCUS       sequence-149254-        7414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To overexpress RIPK1 K627R in Mammalian Cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7414)
  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 7414)
  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-HA-RIPK1 K627R
FEATURES             Location/Qualifiers
     source          1..7414
                     /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"
     CDS             910..936
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3008..3025)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3014..3238
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3284..3712
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3371..3390)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3581..3602
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3721..3741)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3726..4055
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3906..4041
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3968..3987
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4122..4916
                     /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(4176..4195)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4786..4805
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5090..5211
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5127..5146)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5181..5200
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5260..5276)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5260..5276)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5273..5295)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5284..5300
                     /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(5308..5314,5315..5332,5333..5338))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5353..5374
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5491..5508)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5662..6247)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5742..5761)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6418..7209,7210..7278))
                     /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     7041..7060
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7279..7383)
                     /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 gcttggtacc
      901 atggcctcct acccttatga tgtgccagat tatgcctctc ccgaattcat gcaaccagac
      961 atgtccttga atgtcattaa gatgaaatcc agtgacttcc tggagagtgc agaactggac
     1021 agcggaggct ttgggaaggt gtctctgtgt ttccacagaa cccagggact catgatcatg
     1081 aaaacagtgt acaaggggcc caactgcatt gagcacaacg aggccctctt ggaggaggcg
     1141 aagatgatga acagactgag acacagccgg gtggtgaagc tcctgggcgt catcatagag
     1201 gaagggaagt actccctggt gatggagtac atggagaagg gcaacctgat gcacgtgctg
     1261 aaagccgaga tgagtactcc gctttctgta aaaggaagga taattttgga aatcattgaa
     1321 ggaatgtgct acttacatgg aaaaggcgtg atacacaagg acctgaagcc tgaaaatatc
     1381 cttgttgata atgacttcca cattaagatc gcagacctcg gccttgcctc ctttaagatg
     1441 tggagcaaac tgaataatga agagcacaat gagctgaggg aagtggacgg caccgctaag
     1501 aagaatggcg gcaccctcta ctacatggcg cccgagcacc tgaatgacgt caacgcaaag
     1561 cccacagaga agtcggatgt gtacagcttt gctgtagtac tctgggcgat atttgcaaat
     1621 aaggagccat atgaaaatgc tatctgtgag cagcagttga taatgtgcat aaaatctggg
     1681 aacaggccag atgtggatga catcactgag tactgcccaa gagaaattat cagtctcatg
     1741 aagctctgct gggaagcgaa tccggaagct cggccgacat ttcctggcat tgaagaaaaa
     1801 tttaggcctt tttatttaag tcaattagaa gaaagtgtag aagaggacgt gaagagttta
     1861 aagaaagagt attcaaacga aaatgcagtt gtgaagagaa tgcagtctct tcaacttgat
     1921 tgtgtggcag taccttcaag ccggtcaaat tcagccacag aacagcctgg ttcactgcac
     1981 agttcccagg gacttgggat gggtcctgtg gaggagtcct ggtttgctcc ttccctggag
     2041 cacccacaag aagagaatga gcccagcctg cagagtaaac tccaagacga agccaactac
     2101 catctttatg gcagccgcat ggacaggcag acgaaacagc agcccagaca gaatgtggct
     2161 tacaacagag aggaggaaag gagacgcagg gtctcccatg acccttttgc acagcaaaga
     2221 ccttacgaga attttcagaa tacagaggga aaaggcactg cttattccag tgcagccagt
     2281 catggtaatg cagtgcacca gccctcaggg ctcaccagcc aacctcaagt actgtatcag
     2341 aacaatggat tatatagctc acatggcttt ggaacaagac cactggatcc aggaacagca
     2401 ggtcccagag tttggtacag gccaattcca agtcatatgc ctagtctgca taatatccca
     2461 gtgcctgaga ccaactatct aggaaataca cccaccatgc cattcagctc cttgccacca
     2521 acagatgaat ctataaaata taccatatac aatagtactg gcattcagat tggagcctac
     2581 aattatatgg agattggtgg gacgagttca tcactactag acagcacaaa tacgaacttc
     2641 aaagaagagc cagctgctaa gtaccaagct atctttgata ataccactag tctgacggat
     2701 aaacacctgg acccaatcag ggaaaatctg ggaaagcact ggaaaaactg tgcccgtaaa
     2761 ctgggcttca cacagtctca gattgatgaa attgaccatg actatgagcg agatggactg
     2821 aaagaaaggg tttaccagat gctccaaaag tgggtgatga gggaaggcat aaagggagcc
     2881 acggtgggga agctggccca ggcgctccac cagtgttcca ggatcgacct tctgagcagc
     2941 ttgatttacg tcagccagaa ctaactcgag catgcatcta gagggcccgt ttaaacccgc
     3001 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3061 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3121 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3181 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3241 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3301 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3361 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3421 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3481 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3541 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3601 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3661 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3721 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3781 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3841 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3901 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3961 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     4021 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     4081 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     4141 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4201 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4261 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     4321 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4381 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4441 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4501 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4561 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4621 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4681 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4741 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4801 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4861 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4921 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4981 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     5041 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     5101 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5161 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     5221 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     5281 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5341 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5401 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5461 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5521 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5581 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5641 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5701 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5761 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5821 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5881 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5941 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6001 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6061 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6121 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6181 aaccaccgct ggtagcggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6241 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6301 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6361 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6421 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6481 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6541 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6601 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6661 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6721 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6781 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6841 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6901 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6961 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7021 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7081 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7141 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7201 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7261 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7321 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7381 gcgcacattt ccccgaaaag tgccacctga cgtc
//
