LOCUS       sequence-481694-        7690 bp    DNA     circular SYN 01-AUG-2025
DEFINITION  Expression of N-terminal FLAG tagged human gasdermin D.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7690)
  AUTHORS   LaRock DL, Johnson AF, Wilde S, Sands JS, Monteiro MP, LaRock CN
  TITLE     Group A Streptococcus induces GSDMA-dependent pyroptosis in 
            keratinocytes.
  JOURNAL   Nature. 2022 May;605(7910):527-531. doi: 10.1038/s41586-022-04717-x.
            Epub 2022 May 11.
  PUBMED    35545676
REFERENCE   2  (bases 1 to 7690)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-022-04717-x"; journalName: "Nature"; date: 
            "2022-05"; volume: "605"; issue: "7910"; pages: "527-531"
COMMENT     Sequence Label: pCMV-Flag-hGSDMD
FEATURES             Location/Qualifiers
     source          1..7690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1005..1024
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1005..1023
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1029..1163
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1053..1072)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1107..1126
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1815..2609)
                     /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(1926..1945)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2536..2555
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2663..2960)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     2958..2978
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    3002..3624
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     3406..3425
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     CDS             complement(5105..5128)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(5172..5375)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5201..5221)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5376..5755
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(5916..5933)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5916..5932)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5925..5947)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(6073..6528)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6210..6231)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6422..6441
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6555..6659
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6660..6728,6729..7520)
                     /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(6878..6897)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga catgattacg aatttaatac gactcactat
     1021 agggaattga tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     1081 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     1141 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     1201 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg
     1261 attatgatcg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttccagc
     1321 ttggcgagtg ggtggtgagc agctcgccgt cgcgcagcag gcgccagcgc tccatccacg
     1381 cagtgacctc aggcggaaaa cgggaagaca cactcatgag atgcctgcaa gcaattcgtt
     1441 ctgtatcagg cgcaggagcg tcccgtccgg gtcgaccaaa gcggccatcg tgcctcccca
     1501 ctcctgcagt tcgggggcat ggatgcgcgg atagccgctg ctggtttcct ggatgccgac
     1561 ggatttgcac tgccggtaga actccgcgag gtcgtccagc ctcaggcagc agctgaacca
     1621 actcgcgagg ggatcgagcc cggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1681 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1741 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     1801 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     1861 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     1921 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     1981 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     2041 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     2101 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     2161 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     2221 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     2281 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     2341 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     2401 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     2461 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     2521 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     2581 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     2641 gatcccaagc tgatcaattc ctccaaaaaa gcctcctcac tacttctgga atagctcaga
     2701 ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga
     2761 atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt
     2821 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt
     2881 tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     2941 gcctggggac tttccacacc ctaactgaca cacattccac agccaagctg gccgcgtacc
     3001 caattcaaca ggcatctact gagtggaccc aacgcatgag aggacagtgc caagcaagca
     3061 actcaaatgt cccaccggtt gggcatggcc aggtagccta tgctgtgtct ggacgtcctc
     3121 ctgctggtat agttatttta aaatcagaag gacagggaag ggagcagtgg ttcacgcctg
     3181 taatcccagc aatttgggag gccaaggtgg gtagatcacc tgagattagg agttggagac
     3241 cagcctggcc aatatggtga aaccccgtct ctaccaaaaa aacaaaaatt agctgagcct
     3301 ggtcatgcat gcctggaatc ccaacaactc gggaggctga ggcaggagaa tcgcttgaac
     3361 ccaggaggcg gagattgcag tgagccaaga ttgtgccact gcactccagc ttggttccca
     3421 atagaccccg caggccctac aggttgtctt cccaacttgc cccttgctcc ataccacccc
     3481 cctccacccc ataatattat agaaggacac ctagtcagac aaaatgatgc aacttaattt
     3541 tattaggaca aggctggtgg gcactggagt ggcaacttcc agggccagga gaggcactgg
     3601 ggaggggtca cagggatgcc acccgggatc ctctagacta gtggggctcc tggctcagtc
     3661 ctgatagcag tgccagggag gcgtagagtg cacacatgcg gccctgggcc tgcggctccc
     3721 agcacacgtg gggagtgtcc tcccccagct ctaggccaca ctcgtccagc aagacccagg
     3781 ccggtgctcc ttcgccccag ctgttcccca ggagcccggg gggcagggac atggtgctgc
     3841 gctcctgcca cggggcactc tgctccaaga ggctgcccac cagctcgagc ggccccaaca
     3901 gggtctgcga ctccagcgcc tccgccagca gcttgtgctg cgtttcactc agcatggtca
     3961 gtgcccccag caggtagaca acagggatag cgagttccgg caccagcatt ccggaggaca
     4021 acaccaggca ctccaggaca gcacctgctg gaccgtccag gggctccacc ggcccaaggc
     4081 tctggccctg ctccagcgcc tcctccaagg ctcgcagggc cagctggtcc cgcagcaccc
     4141 cctccaggcc ctccagcagc agctggcaca gctctctgtc caaaagctcc agttccttgg
     4201 agatggtctc cacctctgcc cgtaggccct ggaagtcttc agtgaacgcc ccctccgcag
     4261 ggaccccatc tgtcaggaag ttgtggaggc acctcatcat ggagaggcca gagggcagct
     4321 gtggccaggc gccttcgctc gtggaacgct tgtggcctgt cgcgggtggc tggaaggtcc
     4381 tctgcttctt atccgggaag agaaggacgt ccaagtcaga gtcaataacc agctgggcca
     4441 cccggaatgc gagggtgctg cctgagggga tggtgaccgt cttcttctgg ctcagatggc
     4501 cctggccctc accctgcaag cacgtggctc cgggcaggga aaaccggccc gagccctccc
     4561 gcttgtgggt gcgcgtgact tccacctcct tctgtgtctg cagcacctca gtcaccacgt
     4621 acacgttgtc cccgcggctg cgcagctgct gcaggacttt gtgttctggc tgccgcaggt
     4681 gcctctcatg gagcagagtc tgccaggtgt tagggtccac actcagcgag tacacattca
     4741 ttgaggtgct ggagctgtca gacaccgcgg ccccgcctgc gatctttgcc tgtcctgggg
     4801 ctgccagctc cacgctgccc tgtatctgcc catccatggc atcgtagaag tggaagctcc
     4861 tgccacgctg cacgtctggt tccgcggcat ccggctccag gatgtccttg atagacaggt
     4921 tgacacactt ataacggggt ttccagaacc atgagcttga gggcttccta accaccaggc
     4981 agtagggctg gaagccagtg gagctctgca ggctggtcac agggatgaac tccccaccat
     5041 ggtccagctc ctggaccact ctccggacta cccgctcaaa ggccgacccc attgcggccg
     5101 caagcttgtc gtcatcgtct ttgtagtcca tggtagatcg atctgaatta attctgacgg
     5161 ttcactaaac gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc
     5221 gtcaacgggg cggggttatt acgacatttt ggaaagtccc gttgattttg gtgccaaaac
     5281 aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat
     5341 ccacgcccat tggtgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg
     5401 tagatgtact gccaagtagg aaagtcccgt aaggtcatgt actgggcata atgccaggcg
     5461 ggccatttac cgtcattgac gtcaataggg ggcggacttg gcatatgata cacttgatgt
     5521 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct
     5581 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
     5641 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
     5701 tgaactaatg accccgtaat tgattactat taataactag tcaataatca atgtcaacat
     5761 ggcggtcata ttggacatga gccaatataa atgtacatat tatgatatag atacaacgta
     5821 tgcaatggcc aatagccaat attgatttat gctatataac caatgactaa tatggctaat
     5881 tgccaatatt gattcaatgt atagatcagc ttggcactgg ccgtcgtttt acaacgtcgt
     5941 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     6001 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     6061 aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     6121 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     6181 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     6241 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     6301 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     6361 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     6421 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     6481 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     6541 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     6601 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     6661 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     6721 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     6781 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     6841 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     6901 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     6961 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     7021 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     7081 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     7141 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     7201 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     7261 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     7321 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     7381 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     7441 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     7501 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     7561 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     7621 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     7681 aaggatcttc
//
