LOCUS       sequence-221709-        7419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Flag-mRIPK1-S321A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7419)
  AUTHORS   Geng J, Ito Y, Shi L, Amin P, Chu J, Ouchida AT, Mookhtiar AK, Zhao 
            H, Xu D, Shan B, Najafov A, Gao G, Akira S, Yuan J
  TITLE     Regulation of RIPK1 activation by TAK1-mediated phosphorylation 
            dictates apoptosis and necroptosis.
  JOURNAL   Nat Commun. 2017 Aug 25;8(1):359. doi: 10.1038/s41467-017-00406-w.
  PUBMED    28842570
REFERENCE   2  (bases 1 to 7419)
  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: "Nat 
            Commun."; date: "2017-08-25"; volume: "8(1):359. doi"; pages: " 
            10.1038/s41467-017-00406-w"
COMMENT     Sequence Label: pcDNA3.1-Flag-mRIPK1-S321A
FEATURES             Location/Qualifiers
     source          1..7419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             333..356
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2358..2376)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2359..2376)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2396..2413)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2402..2626
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2672..3100
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2759..2778)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2969..2990
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3109..3129)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3114..3443
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3294..3429
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3356..3375
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3510..4304
                     /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(3564..3583)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4174..4193
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4478..4599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4515..4534)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4569..4588
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4648..4664)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4648..4664)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4661..4683)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4672..4688
                     /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(4696..4702,4703..4720,4721..4726))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4741..4762
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4879..4896)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5050..5638)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5130..5149)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5809..6600,6601..6669))
                     /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     6432..6451
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6670..6774)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6849..6868)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7040..7419
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctt ggtaccgagc tcggatccac
      301 tagtaacggc cgccagtgtg ctggaattca tggactacaa agacgatgac gacaagggca
      361 tgcaaccaga catgtccttg gacaatatta agatggcatc cagtgacctg ctggagaaga
      421 cagacctaga cagcggaggc ttcgggaagg tgtccttgtg ttaccacaga agccatggat
      481 ttgtcatcct gaaaaaagta tacacagggc ccaaccgcgc tgagtacaat gaggttctct
      541 tggaagaggg gaagatgatg cacagactga gacacagtcg agtggtgaag ctactgggca
      601 tcatcataga agaagggaac tattcgctgg tgatggagta catggagaag ggcaacctga
      661 tgcacgtgct aaagacccag atagatgtcc cactttcatt gaaaggaagg ataatcgtgg
      721 aggccataga aggcatgtgc tacttacatg acaaaggtgt gatacacaag gacctgaagc
      781 ctgagaatat cctcgttgat cgtgactttc acattaagat agccgatctt ggtgtggctt
      841 cctttaagac atggagcaaa ctgactaagg agaaagacaa caagcagaaa gaagtgagca
      901 gcaccactaa gaagaacaat ggtggtaccc tttactacat ggcacccgaa cacctgaatg
      961 acatcaatgc aaagcccacg gagaagtcgg acgtgtacag ctttggcatt gtcctttggg
     1021 caatatttgc aaaaaaggag ccctatgaga atgtcatctg tactgagcag ttcgtgatct
     1081 gcataaaatc tgggaacagg ccaaatgtag aggaaatcct tgagtactgt ccaagggaga
     1141 tcatcagcct catggagcgg tgctggcagg cgatcccaga agacaggcca acatttcttg
     1201 gcattgaaga agaatttagg cctttttact taagtcattt tgaagaatat gtagaagagg
     1261 atgtggcaag tttaaagaaa gagtatccag atcaaagccc agtgctgcag agaatgtttg
     1321 cactccagca tgactgtgta cccttacctc cgagcaggtc aaattcagaa caacctggat
     1381 cgctgcacag ttcccagggg ctccagatgg gtcctgtgga ggagtcctgg ttttcttcct
     1441 ccccagagta cccacaggac gagaatgatc gcagtgtgca ggctaagctg caagaggaag
     1501 ccagctatca tgcttttgga atatttgcag agaaacagac aaaaccgcag ccaaggcaga
     1561 atgaggctta caacagagag gaggaaagga aacgaagggt ctctcatgac ccctttgcac
     1621 agcagagagc tcgtgagaat attaagagtg caggagcaag aggtcattct gatcccagca
     1681 caacgagtcg tggaattgca gtgcaacagc tgtcatggcc agccacccaa acagtttgga
     1741 acaatggatt gtataatcag catggatttg gaactatagg tacaggagtt tggtatccgc
     1801 caaatctaag ccaaatgtat agtacttata aaactccagt gccagagacc aacataccgg
     1861 gaagcacacc caccatgcca tacttctctg ggccagtagc agatgacctc ataaaatata
     1921 ctatattcaa tagttctggt attcagattg gaaaccacaa ttatatggat gttggactga
     1981 attcacaacc accaaacaat acttgcaaag aagagtcgac ttccagacac caagccatct
     2041 ttgataacac cactagtctg actgatgaac acctgaaccc tatcagggaa aacctgggaa
     2101 ggcagtggaa aaactgtgcc cgcaagctgg gcttcactga gtctcagatc gatgaaatcg
     2161 accatgacta tgaaagagat ggactgaaag agaaagttta ccaaatgctt cagaagtggc
     2221 tgatgcggga aggcaccaaa ggggccacag tgggaaagtt ggcccaggca cttcaccaat
     2281 gttgcaggat agacctgctg aaccacttga ttcgtgccag ccagagctaa ctcgagcatg
     2341 catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg
     2401 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2461 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2521 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2581 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2641 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2701 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2761 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2821 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2881 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2941 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3001 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3061 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     3121 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3181 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3241 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3301 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3361 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     3421 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     3481 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3541 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3601 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3661 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3721 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3781 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3841 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3901 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3961 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4021 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4081 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4141 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4201 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4261 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4321 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4381 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4441 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     4501 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4561 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     4621 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4681 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4741 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4801 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4861 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4921 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4981 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5041 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5101 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5161 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5221 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5281 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5341 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5401 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5461 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5521 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5581 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5641 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5701 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5761 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5821 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5881 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5941 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6001 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6061 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6121 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6181 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6241 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6301 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6361 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6421 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6481 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6541 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6601 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6661 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6721 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6781 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     6841 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     6901 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     6961 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     7021 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     7081 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     7141 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     7201 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     7261 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     7321 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     7381 tggcagtaca tctacgtatt agtcatcgct attaccatg
//
