LOCUS       sequence-266231-        7066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Gsdmd-NT mutant in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7066)
  AUTHORS   Liu X, Zhang Z, Ruan J, Pan Y, Magupalli VG, Wu H, Lieberman J
  TITLE     Inflammasome-activated gasdermin D causes pyroptosis by forming 
            membrane pores.
  JOURNAL   Nature. 2016 Jul 6;535(7610):153-8. doi: 10.1038/nature18629.
  PUBMED    27383986
REFERENCE   2  (bases 1 to 7066)
  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"; journalName: 
            "Nature"; date: "2016-07-6- 6"; volume: "535"; issue: "7610"; pages:
            "153-8"
COMMENT     Sequence Label: Flag-Gsdmd-NT C192A
FEATURES             Location/Qualifiers
     source          1..7066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     131..153
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     145..162
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     146..162
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        323..702
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        703..906
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     857..877
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             950..973
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1830..2452
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2029..2048)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2476..2496)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2494..2791
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2845..3639
                     /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(2899..2918)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3509..3528
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4291..4425
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4328..4347)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4382..4401
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4430..4449)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4431..4449)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4463..4479)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4463..4479)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4476..4498)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4487..4503
                     /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(4511..4517,4518..4535,4536..4541))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4556..4577
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4694..4711)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4865..5453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4945..4964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5624..6415,6416..6484))
                     /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     6247..6266
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6485..6589)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6616..7044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6703..6722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6913..6934
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aatttccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
       61 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
      121 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct gatctataca
      181 ttgaatcaat attggcaatt agccatatta gtcattggtt atatagcata aatcaatatt
      241 ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta tattggctca
      301 tgtccaatat gaccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt
      361 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      421 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      481 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      541 actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc
      601 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct
      661 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      721 tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      781 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat
      841 aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      901 agagctcgtt tagtgaaccg tcagaattaa ttcagatcga tctaccatgg actacaaaga
      961 cgatgacgac aagcttgcgg ccgcaatgcc atcggccttt gagaaagtgg tcaagaatgt
     1021 gatcaaggag gtaagcggca gcagaggcga tctcattccg gtggacagcc tgcggaactc
     1081 caccagcttc aggccctact gccttctgaa caggaaattt tcaagctcaa ggttctggaa
     1141 accccgttat tcatgtgtca acctgtcaat caaggacatc ctggagccca gtgctccaga
     1201 accagaaccg gagtgttttg gctccttcaa agtctctgat gtcgtcgatg ggaacattca
     1261 gggcagagtg atgttgtcag gcatgggaga agggaaaatt tctggtgggg ctgcagtgtc
     1321 tgacagttcc agtgcctcca tgaatgtgtg tatactgcgt gtgactcaga agacctggga
     1381 gaccatgcag catgaaaggc accttcagca gcctgagaac aaaatcctgc aacagcttcg
     1441 gagtcgtggg gatgacctgt ttgtggtgac cgaggtgctg cagacaaagg aggaagtgca
     1501 gatcactgag gtccacagcc aagagggctc aggccagttt acgctgcctg gagctttagc
     1561 cttgaagggt gaaggcaagg gccaccaaag ccggaagaag atggtgacca ttcctgcagg
     1621 cagcatcctg gcattccgag tggcccaact gcttattggc tctaaatggg atatccttct
     1681 cgtctcagat gagaaacaga ggacctttga gccctcctca ggtgacagaa aagcagtggg
     1741 ccagaggcac catggcctca atgtgcttgc tgcgctttgt tccatcggaa agcagctcag
     1801 tctcctgtca gattagtcta gaggatcccg ggtggcatcc ctgtgacccc tccccagtgc
     1861 ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa
     1921 gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
     1981 tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac
     2041 caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag
     2101 cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca
     2161 gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca
     2221 actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
     2281 gaaccactgc tcccttccct gtccttctga ttttaaaata actataccag caggaggacg
     2341 tccagacaca gcataggcta cctggccatg cccaaccggt gggacatttg agttgcttgc
     2401 ttggcactgt cctctcatgc gttgggtcca ctcagtagat gcctgttgaa ttgggtacgc
     2461 ggccagcttg gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2521 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2581 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2641 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2701 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     2761 tattccagaa gtagtgagga ggcttttttg gaggaattga tcagcttggg atctgatcaa
     2821 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     2881 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     2941 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3001 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3061 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3121 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3181 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3241 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     3301 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     3361 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     3421 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     3481 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     3541 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     3601 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     3661 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     3721 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     3781 gggatctcat gctggagttc ttcgcccacc ccgggctcga tcccctcgcg agttggttca
     3841 gctgctgcct gaggctggac gacctcgcgg agttctaccg gcagtgcaaa tccgtcggca
     3901 tccaggaaac cagcagcggc tatccgcgca tccatgcccc cgaactgcag gagtggggag
     3961 gcacgatggc cgctttggtc gacccggacg ggacgctcct gcgcctgata cagaacgaat
     4021 tgcttgcagg catctcatga gtgtgtcttc ccgttttccg cctgaggtca ctgcgtggat
     4081 ggagcgctgg cgcctgctgc gcgacggcga gctgctcacc acccactcgc caagctggaa
     4141 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgccgatcat aatcagccat
     4201 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     4261 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     4321 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4381 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcaattc cctatagtga
     4441 gtcgtattaa attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4501 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4561 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4621 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4681 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4741 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4801 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4861 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4921 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4981 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5041 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5101 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5161 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5221 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5281 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5341 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5401 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5461 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5521 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5581 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5641 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5701 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5761 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5821 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5881 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5941 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6001 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6061 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6121 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6181 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6241 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6301 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6361 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6421 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6481 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6541 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6601 gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     6661 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     6721 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     6781 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     6841 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     6901 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     6961 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     7021 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttac
//
