LOCUS       sequence-430564-        6203 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Gasdermin1-270 is a pore-forming GSDMD formed by SARS-CoV-2 3CLpro 
            cleavage. Transfection in HEK293 cells showed GSDMD 1-270 pores 
            secrete 3CLpro, nucleocapsid, and in excess, kill cells by 
            pyroptosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6203)
  TITLE     Gasdermin-D/E cleavage and pore formation mediate unconventional 
            protein secretion and extracellular activity of SARS-CoV-2 3CLpro 
            (nsp5)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6203)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pcDNA3.1-GSDMD_1-270
FEATURES             Location/Qualifiers
     source          1..6203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      935..944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1751..1774
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1794..1811)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1800..2024
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2070..2498
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2157..2176)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2367..2388
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2507..2527)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2512..2841
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2692..2827
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2754..2773
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2908..3702
                     /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(2962..2981)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3572..3591
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3876..3997
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3913..3932)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3967..3986
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4046..4062)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4046..4062)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4059..4081)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4070..4086
                     /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(4094..4100,4101..4118,4119..4124))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4139..4160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4277..4294)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4448..5036)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4528..4547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5207..5998,5999..6067))
                     /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     5830..5849
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6068..6172)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccgccacc atggggtcgg cctttgagcg
      961 ggtagtccgg agagtggtcc aggagctgga ccatggtggg gagttcatcc ctgtgaccag
     1021 cctgcagagc tccactggct tccagcccta ctgcctggtg gttaggaagc cctcaagctc
     1081 atggttctgg aaaccccgtt ataagtgtgt caacctgtct atcaaggaca tcctggagcc
     1141 ggatgccgcg gaaccagacg tgcagcgtgg caggagcttc cacttctacg atgccatgga
     1201 tgggcagata cagggcagcg tggagctggc agccccagga caggcaaaga tcgcaggcgg
     1261 ggccgcggtg tctgacagct ccagcacctc aatgaatgtg tactcgctga gtgtggaccc
     1321 taacacctgg cagactctgc tccatgagag gcacctgcgg cagccagaac acaaagtcct
     1381 gcagcagctg cgcagccgcg gggacaacgt gtacgtggtg actgaggtgc tgcagacaca
     1441 gaaggaggtg gaagtcacgc gcacccacaa gcgggagggc tcgggccggt tttccctgcc
     1501 cggagccacg tgcttgcagg gtgagggcca gggccatctg agccagaaga agacggtcac
     1561 catcccctca ggcagcaccc tcgcattccg ggtggcccag ctggttattg actctgactt
     1621 ggacgtcctt ctcttcccgg ataagaagca gaggaccttc cagccacccg cgacaggcca
     1681 caagcgttcc acgagcgaag gcgcctggcc acagctgccc tctggcctct ccatgatgag
     1741 gtgcctccac gattacaagg atgacgacga taagtgataa acccgctgat cagcctcgac
     1801 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1861 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1921 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1981 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2041 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2101 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2161 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2221 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2281 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2341 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2401 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2461 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     2521 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2581 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2641 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2701 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2761 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     2821 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     2881 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     2941 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3001 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3061 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     3121 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3181 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3241 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3301 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3361 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3421 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     3481 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     3541 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     3601 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3661 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3721 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     3781 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3841 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3901 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3961 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     4021 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4081 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4141 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4201 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4261 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4321 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4381 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4441 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4501 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4561 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4621 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4681 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4741 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4801 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4861 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4921 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4981 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5041 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5101 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5161 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5221 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5281 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5341 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5401 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5461 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5521 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5581 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5641 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5701 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5761 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5821 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5881 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5941 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6001 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6061 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6121 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6181 cccgaaaagt gccacctgac gtc
//
