LOCUS       sequence-444268-        6845 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of mutant Gasdermin D_Q29A_H270A (GSDMD_Q29A_H270A) in 
            transfected mammalian cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6845)
  TITLE     Release and activity of SARS-CoV-2 main protease (3CLpro) in the 
            extracellular environment by gasdermin-D/E pore formation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6845)
  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            "Unpublished"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_Q29A_H270A
FEATURES             Location/Qualifiers
     source          1..6845
                     /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             2393..2416
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2436..2453)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2442..2666
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2712..3140
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2799..2818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3009..3030
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3149..3169)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3154..3483
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3334..3469
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3396..3415
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3550..4344
                     /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(3604..3623)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4214..4233
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4518..4639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4555..4574)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4609..4628
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4688..4704)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4688..4704)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4701..4723)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4712..4728
                     /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(4736..4742,4743..4760,4761..4766))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4781..4802
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4919..4936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5090..5678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5170..5189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5849..6640,6641..6709))
                     /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     6472..6491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6710..6814)
                     /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 cctggcgagc 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 gtgcctcgcc aacttcctga cagatggggt ccctgcggag ggggcgttca ctgaagactt
     1801 ccagggccta cgggcagagg tggagaccat ctccaaggaa ctggagcttt tggacagaga
     1861 gctgtgccag ctgctgctgg agggcctgga gggggtgctg cgggaccagc tggccctgcg
     1921 agccttggag gaggcgctgg agcagggcca gagccttggg ccggtggagc ccctggacgg
     1981 tccagcaggt gctgtcctgg agtgcctggt gttgtcctcc ggaatgctgg tgccggaact
     2041 cgctatccct gttgtctacc tgctgggggc actgaccatg ctgagtgaaa cgcagcacaa
     2101 gctgctggcg gaggcgctgg agtcgcagac cctgttgggg ccgctcgagc tggtgggcag
     2161 cctcttggag cagagtgccc cgtggcagga gcgcagcacc atgtccctgc cccccgggct
     2221 cctggggaac agctggggcg aaggagcacc ggcctgggtc ttgctggacg agtgtggcct
     2281 agagctgggg gaggacactc cccacgtgtg ctgggagccg caggcccagg gccgcatgtg
     2341 tgcactctac gcctccctgg cactgctatc aggactgagc caggagcccc acgattacaa
     2401 ggatgacgac gataagtgat aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     2461 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     2521 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     2581 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     2641 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     2701 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2761 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2821 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2881 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2941 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3001 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3061 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3121 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3181 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3241 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3301 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3361 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3421 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3481 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     3541 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     3601 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     3661 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     3721 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     3781 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     3841 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     3901 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     3961 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     4021 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     4081 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4141 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4201 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4261 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4321 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     4381 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4441 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4501 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4561 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4621 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     4681 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4741 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4801 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4861 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4921 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4981 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5041 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5101 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5161 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5221 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5281 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5341 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5401 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5461 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5521 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5581 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5641 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5701 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5761 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5821 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5881 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5941 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6001 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6061 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6121 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6181 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6241 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6301 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6361 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6421 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6481 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6541 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6601 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6661 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6721 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6781 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6841 acgtc
//
