LOCUS       sequence-479869-        7726 bp    DNA     circular SYN 24-JUL-2025
DEFINITION  Expression of N-terminal FLAG tagged human gasdermin E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7726)
  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 7726)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 24, 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-hGSDME
FEATURES             Location/Qualifiers
     source          1..7726
                     /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(5141..5164)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(5208..5411)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5237..5257)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5412..5791
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(5952..5968)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5961..5983)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(6109..6564)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6246..6267)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6458..6477
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6591..6695
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6696..6764,6765..7556)
                     /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(6914..6933)
                     /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 ctctagatca tgaatgttct ctgcctaaag
     3661 cacagagtcc attcagggtt atacaaagaa gcagtgggaa gactttagag tccttcagaa
     3721 tgacagcttt cacagatgac ttcagtctct ccagactaat gtcagctgag gcaaacaagc
     3781 gctgcacaat cccaaacctt tctgtatctt tcaggggagt caaggttggg tcttcaagat
     3841 cagatactcc atcatcagac agagcacgaa gcaagtggca cagtgtggga atgatctgga
     3901 gtttgcagca agtgcccagc agagctgctg cgctatctgg catttctgcg agggcactga
     3961 ccaagaagta ggctgtcata aacagctgct tgctgcctgc atcctcgggg cccggacacc
     4021 caccctgtaa gctgcacccc accagctgca ggaaggccac aaggtcctgc tgctgccggg
     4081 gcttcagctc ccccagcacc gccactgtgg gcgagaggcc gctgaccagg tcatcgcaca
     4141 ctggttccag gaccatgagt agttcatcat caaataggac cgcctggaag atgtcactca
     4201 aagctgtctg ttgtggctca ggcagctccg caaatggatg gaaattcctc tccaggagca
     4261 gggtcgcttg ctttaaaaca cttaatggtc catcctggga agatatccca tgcgcagcat
     4321 ctggcatgtc tatgaatgca aactctcgaa agaccagggg gtccaggtag acagagtcaa
     4381 ttctcttctt gttctcgaag ccaccttgct tccctcggag aaggcagaac tcgaactggc
     4441 cgtccagttt cacgtataac tcaatgacac cgtaggcaat ggtggtggca gctgggatct
     4501 ccagcaccac gttggagtcc ttggtgacat tcccatcctc cgtcgctgac acctgcaccg
     4561 tcttggtctg gatgcccacg atgccaccac acttctcctc gacctgcatg tgctcagaga
     4621 tcacacactt ctgcatcgtc gtgatcttct gtgtcaaaac gcacaggacc tcattccttc
     4681 cttccagcac ctgctggagc acagggtttc tcagatttat tgttctctcg gcagagtctc
     4741 tgatgagctg ctgcaaatcc acctcctgct tcctcagggt tccaaatgaa gactggctct
     4801 ctacgcggct gctgcccccc aggttcagct tgaccttccc cagtgcagtc tccagggttc
     4861 cactcacgtg gtttgcaaac ttgccctcgt atttcacaaa gtccgactcc acgaccactg
     4921 gactcggaaa ttggtcttct atgagtacat cgccaagggt gagggataaa aactggtact
     4981 tgggtctctg ccagcaccag aatctcttct tttttgtcac cagacttaga agctgtaact
     5041 tatcagagtc attcagattt gatactgcaa tcaggtcacc atcagcatca acttctctaa
     5101 gaaaattcct ggttgctttg gcaaacattg cggccgcaag cttgtcgtca tcgtctttgt
     5161 agtccatggt agatcgatct gaattaattc tgacggttca ctaaacgagc tctgcttata
     5221 tagacctccc accgtacacg cctaccgccc atttgcgtca acggggcggg gttattacga
     5281 cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg
     5341 tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattggt gtactgccaa
     5401 aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag
     5461 tcccgtaagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca
     5521 atagggggcg gacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt
     5581 aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac
     5641 gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc
     5701 gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat
     5761 tactattaat aactagtcaa taatcaatgt caacatggcg gtcatattgg acatgagcca
     5821 atataaatgt acatattatg atatagatac aacgtatgca atggccaata gccaatattg
     5881 atttatgcta tataaccaat gactaatatg gctaattgcc aatattgatt caatgtatag
     5941 atcagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     6001 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     6061 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa attgtaagcg
     6121 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     6181 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     6241 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     6301 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     6361 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     6421 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     6481 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     6541 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     6601 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     6661 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     6721 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6781 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6841 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6901 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6961 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     7021 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     7081 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     7141 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     7201 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     7261 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     7321 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     7381 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     7441 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     7501 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     7561 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     7621 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     7681 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
