LOCUS       sequence-221699-        7419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Flag-mRIPK1-S321E.
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-S321E
FEATURES             Location/Qualifiers
     source          1..7419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             713..736
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2738..2756)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2739..2756)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2776..2793)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2782..3006
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3052..3480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3139..3158)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3349..3370
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3489..3509)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3494..3823
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3674..3809
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3736..3755
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3890..4684
                     /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(3944..3963)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4554..4573
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4858..4979
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4895..4914)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4949..4968
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5028..5044)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5028..5044)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5041..5063)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5052..5068
                     /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(5076..5082,5083..5100,5101..5106))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5121..5142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5259..5276)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5430..6018)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5510..5529)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6189..6980,6981..7049))
                     /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     6812..6831
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7050..7154)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7229..7248)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattca tggactacaa
      721 agacgatgac gacaagggca tgcaaccaga catgtccttg gacaatatta agatggcatc
      781 cagtgacctg ctggagaaga cagacctaga cagcggaggc ttcgggaagg tgtccttgtg
      841 ttaccacaga agccatggat ttgtcatcct gaaaaaagta tacacagggc ccaaccgcgc
      901 tgagtacaat gaggttctct tggaagaggg gaagatgatg cacagactga gacacagtcg
      961 agtggtgaag ctactgggca tcatcataga agaagggaac tattcgctgg tgatggagta
     1021 catggagaag ggcaacctga tgcacgtgct aaagacccag atagatgtcc cactttcatt
     1081 gaaaggaagg ataatcgtgg aggccataga aggcatgtgc tacttacatg acaaaggtgt
     1141 gatacacaag gacctgaagc ctgagaatat cctcgttgat cgtgactttc acattaagat
     1201 agccgatctt ggtgtggctt cctttaagac atggagcaaa ctgactaagg agaaagacaa
     1261 caagcagaaa gaagtgagca gcaccactaa gaagaacaat ggtggtaccc tttactacat
     1321 ggcacccgaa cacctgaatg acatcaatgc aaagcccacg gagaagtcgg acgtgtacag
     1381 ctttggcatt gtcctttggg caatatttgc aaaaaaggag ccctatgaga atgtcatctg
     1441 tactgagcag ttcgtgatct gcataaaatc tgggaacagg ccaaatgtag aggaaatcct
     1501 tgagtactgt ccaagggaga tcatcagcct catggagcgg tgctggcagg cgatcccaga
     1561 agacaggcca acatttcttg gcattgaaga agaatttagg cctttttact taagtcattt
     1621 tgaagaatat gtagaagagg atgtggcaag tttaaagaaa gagtatccag atcaaagccc
     1681 agtgctgcag agaatgtttg aactccagca tgactgtgta cccttacctc cgagcaggtc
     1741 aaattcagaa caacctggat cgctgcacag ttcccagggg ctccagatgg gtcctgtgga
     1801 ggagtcctgg ttttcttcct ccccagagta cccacaggac gagaatgatc gcagtgtgca
     1861 ggctaagctg caagaggaag ccagctatca tgcttttgga atatttgcag agaaacagac
     1921 aaaaccgcag ccaaggcaga atgaggctta caacagagag gaggaaagga aacgaagggt
     1981 ctctcatgac ccctttgcac agcagagagc tcgtgagaat attaagagtg caggagcaag
     2041 aggtcattct gatcccagca caacgagtcg tggaattgca gtgcaacagc tgtcatggcc
     2101 agccacccaa acagtttgga acaatggatt gtataatcag catggatttg gaactatagg
     2161 tacaggagtt tggtatccgc caaatctaag ccaaatgtat agtacttata aaactccagt
     2221 gccagagacc aacataccgg gaagcacacc caccatgcca tacttctctg ggccagtagc
     2281 agatgacctc ataaaatata ctatattcaa tagttctggt attcagattg gaaaccacaa
     2341 ttatatggat gttggactga attcacaacc accaaacaat acttgcaaag aagagtcgac
     2401 ttccagacac caagccatct ttgataacac cactagtctg actgatgaac acctgaaccc
     2461 tatcagggaa aacctgggaa ggcagtggaa aaactgtgcc cgcaagctgg gcttcactga
     2521 gtctcagatc gatgaaatcg accatgacta tgaaagagat ggactgaaag agaaagttta
     2581 ccaaatgctt cagaagtggc tgatgcggga aggcaccaaa ggggccacag tgggaaagtt
     2641 ggcccaggca cttcaccaat gttgcaggat agacctgctg aaccacttga ttcgtgccag
     2701 ccagagctaa ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta
     2761 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2821 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2881 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2941 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3001 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3061 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3121 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3181 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3241 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3301 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3361 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3421 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3481 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3541 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3601 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3661 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3721 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     3781 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     3841 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3901 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3961 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4021 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4081 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4141 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4201 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4261 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4321 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4381 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4441 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4501 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4561 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4621 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4681 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4741 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4801 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     4861 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4921 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4981 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5041 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5101 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5161 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5221 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5281 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5341 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5401 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5461 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5521 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5581 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5641 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5701 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5761 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5821 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5881 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5941 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6001 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6061 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6121 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6181 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6241 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6301 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6361 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6421 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6481 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6541 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6601 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6661 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6721 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6781 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6841 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6901 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6961 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7021 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7081 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7141 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     7201 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7261 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     7321 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     7381 ttgcgctgct tcgcgatgta cgggccagat atacgcgtt
//
