LOCUS       sequence-221704-        6477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Liver-specific adeno-associated viral delivery and mammalian 
            expression of Flag-mRIPK1-S321A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6477)
  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 6477)
  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: pAAV2-TBG-FLAG-mRIPK1-S321A
FEATURES             Location/Qualifiers
     source          1..6477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        332..741
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin 
                     gene (Yan et al., 2012)"
     intron          879..1011
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     994..1013
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1056..1074
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1100..1123
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3166..3185)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3231..3286
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   3379..3508
                     /label=AAV2 ITR
     primer_bind     complement(3527..3544)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3527..3543)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3536..3558)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3685..4140
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3772..3791)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3982..4003
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4166..4270
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4271..4339,4340..5131)
                     /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     complement(4489..4508)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5302..5890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5791..5810
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6178..6199
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6214..6219,6220..6237,6238..6244)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6252..6268
                     /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)."
     primer_bind     6257..6279
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6276..6292
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6276..6292
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   6330..6459
                     /label=AAV2 ITR
ORIGIN
        1 cgccatgcta cttatctacc agggtaatgg ggatcctcta gaactatagc tagaattcgc
       61 ccttaagcta gcaggttaat ttttaaaaag cagtcaaaag tccaagtggc ccttggcagc
      121 atttactctc tctgtttgct ctggttaata atctcaggag cacaaacatt ccagatccag
      181 gttaattttt aaaaagcagt caaaagtcca agtggccctt ggcagcattt actctctctg
      241 tttgctctgg ttaataatct caggagcaca aacattccag atccggcgcg ccagggctgg
      301 aagctacctt tgacatcatt tcctctgcga atgcatgtat aatttctaca gaacctatta
      361 gaaaggatca cccagcctct gcttttgtac aactttccct taaaaaactg ccaattccac
      421 tgctgtttgg cccaatagtg agaacttttt cctgctgcct cttggtgctt ttgcctatgg
      481 cccctattct gcctgctgaa gacactcttg ccagcatgga cttaaacccc tccagctctg
      541 acaatcctct ttctcttttg ttttacatga agggtctggc agccaaagca atcactcaaa
      601 gttcaaacct tatcattttt tgctttgttc ctcttggcct tggttttgta catcagcttt
      661 gaaaatacca tcccagggtt aatgctgggg ttaatttata actaagagtg ctctagtttt
      721 gcaatacagg acatgctata aaaatggaaa gatgttgctt tctgagagac agctttattg
      781 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
      841 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
      901 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
      961 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1021 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     1081 tcgagaattc acgcgtatgg actacaaaga cgatgacgac aagggcatgc aaccagacat
     1141 gtccttggac aatattaaga tggcatccag tgacctgctg gagaagacag acctagacag
     1201 cggaggcttc gggaaggtgt ccttgtgtta ccacagaagc catggatttg tcatcctgaa
     1261 aaaagtatac acagggccca accgcgctga gtacaatgag gttctcttgg aagaggggaa
     1321 gatgatgcac agactgagac acagtcgagt ggtgaagcta ctgggcatca tcatagaaga
     1381 agggaactat tcgctggtga tggagtacat ggagaagggc aacctgatgc acgtgctaaa
     1441 gacccagata gatgtcccac tttcattgaa aggaaggata atcgtggagg ccatagaagg
     1501 catgtgctac ttacatgaca aaggtgtgat acacaaggac ctgaagcctg agaatatcct
     1561 cgttgatcgt gactttcaca ttaagatagc cgatcttggt gtggcttcct ttaagacatg
     1621 gagcaaactg actaaggaga aagacaacaa gcagaaagaa gtgagcagca ccactaagaa
     1681 gaacaatggt ggtacccttt actacatggc acccgaacac ctgaatgaca tcaatgcaaa
     1741 gcccacggag aagtcggacg tgtacagctt tggcattgtc ctttgggcaa tatttgcaaa
     1801 aaaggagccc tatgagaatg tcatctgtac tgagcagttc gtgatctgca taaaatctgg
     1861 gaacaggcca aatgtagagg aaatccttga gtactgtcca agggagatca tcagcctcat
     1921 ggagcggtgc tggcaggcga tcccagaaga caggccaaca tttcttggca ttgaagaaga
     1981 atttaggcct ttttacttaa gtcattttga agaatatgta gaagaggatg tggcaagttt
     2041 aaagaaagag tatccagatc aaagcccagt gctgcagaga atgtttgcac tccagcatga
     2101 ctgtgtaccc ttacctccga gcaggtcaaa ttcagaacaa cctggatcgc tgcacagttc
     2161 ccaggggctc cagatgggtc ctgtggagga gtcctggttt tcttcctccc cagagtaccc
     2221 acaggacgag aatgatcgca gtgtgcaggc taagctgcaa gaggaagcca gctatcatgc
     2281 ttttggaata tttgcagaga aacagacaaa accgcagcca aggcagaatg aggcttacaa
     2341 cagagaggag gaaaggaaac gaagggtctc tcatgacccc tttgcacagc agagagctcg
     2401 tgagaatatt aagagtgcag gagcaagagg tcattctgat cccagcacaa cgagtcgtgg
     2461 aattgcagtg caacagctgt catggccagc cacccaaaca gtttggaaca atggattgta
     2521 taatcagcat ggatttggaa ctataggtac aggagtttgg tatccgccaa atctaagcca
     2581 aatgtatagt acttataaaa ctccagtgcc agagaccaac ataccgggaa gcacacccac
     2641 catgccatac ttctctgggc cagtagcaga tgacctcata aaatatacta tattcaatag
     2701 ttctggtatt cagattggaa accacaatta tatggatgtt ggactgaatt cacaaccacc
     2761 aaacaatact tgcaaagaag agtcgacttc cagacaccaa gccatctttg ataacaccac
     2821 tagtctgact gatgaacacc tgaaccctat cagggaaaac ctgggaaggc agtggaaaaa
     2881 ctgtgcccgc aagctgggct tcactgagtc tcagatcgat gaaatcgacc atgactatga
     2941 aagagatgga ctgaaagaga aagtttacca aatgcttcag aagtggctga tgcgggaagg
     3001 caccaaaggg gccacagtgg gaaagttggc ccaggcactt caccaatgtt gcaggataga
     3061 cctgctgaac cacttgattc gtgccagcca gagctaaacg cgtggtacct ctagagtcga
     3121 cccgggcggc ctcgaggacg gggtgaacta cgcctgagga tccgatcttt ttccctctgc
     3181 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     3241 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaagcaattc
     3301 gttgatctga atttcgacca cccataatac ccattaccct ggtagataag tagcatggcg
     3361 ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     3421 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     3481 cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc taattcactg gccgtcgttt
     3541 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     3601 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     3661 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3721 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3781 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3841 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3901 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3961 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     4021 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     4081 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     4141 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4201 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4261 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4321 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4381 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4441 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4501 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4561 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4621 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4681 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4741 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4801 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4861 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4921 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4981 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5041 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5101 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5161 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5221 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5281 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5341 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5401 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5461 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5521 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5581 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5641 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5701 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5761 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5821 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5881 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5941 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     6001 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     6061 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     6121 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     6181 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     6241 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     6301 cgccagattt aattaaggcc ttaattaggc tgcgcgctcg ctcgctcact gaggccgccc
     6361 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
     6421 gcagagaggg agtggccaac tccatcacta ggggttcctt gtagttaatg attaacc
//
