LOCUS       sequence-332581-        7087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of CARD8-FLAG that cannot bind DPP9 due to a 
            mutation at site I (E274R) with an additional autoprocessing 
            mutation (S297A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7087)
  AUTHORS   Sharif H, Hollingsworth LR, Griswold AR, Hsiao JC, Wang Q, 
            Bachovchin DA, Wu H
  TITLE     Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome 
            formation by sequestering its active C-terminal fragment.
  JOURNAL   Immunity. 2021 Jul 13;54(7):1392-1404.e10. doi: 
            10.1016/j.immuni.2021.04.024. Epub 2021 May 20.
  PUBMED    34019797
REFERENCE   2  (bases 1 to 7087)
  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: 
            "10.1016/j.immuni.2021.04.024"; journalName: "Immunity"; date: 
            "2021-07-13- 13"; volume: "54"; issue: "7"; pages: "1392-1404.e10"
COMMENT     Sequence Label: pcDNA3.1 LIC 6A CARD8-FLAG E274R + S297A
FEATURES             Location/Qualifiers
     source          1..7087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(2310..2334)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2342..2365
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2406..2424)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2407..2424)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2444..2461)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2450..2674
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2720..3148
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2807..2826)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3017..3038
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3157..3177)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3162..3491
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3342..3477
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3404..3423
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3558..4352
                     /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(3612..3631)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4222..4241
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4526..4647
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4563..4582)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4617..4636
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4696..4712)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4696..4712)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4709..4731)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4720..4736
                     /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(4744..4750,4751..4768,4769..4774))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4789..4810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4927..4944)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5098..5686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5178..5197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5857..6648,6649..6717))
                     /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     6480..6499
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6718..6822)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6897..6916)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccct acttccaatc caatgcaatg gaaaaaaagg agtgtccaga
      721 aaagagtagc agcagtgagg aagagctgcc gagacgggac agtggatcca gtaggaacat
      781 agatgcatcc aaactcatta gactacaagg atcacggaaa ctgttggttg acaatagcat
      841 acgggaactg caatacacaa aaactggaat tttttttcag gctgaggcct gtgtgacaaa
      901 tgatacggta tacagggagc taccctgtgt ttctgagacc ctttgtgaca tctcacattt
      961 tttccaagaa gatgatgaga cagaggcaga gccattattg ttccgtgctg ttcctgagtg
     1021 tcaactatct gggggggaca ttcccagtgt atcagaagag caggaatctt cagagggaca
     1081 agattcagga gacatttgct cagaagagaa tcaaatagtt tcctcttatg cttctaaagt
     1141 ctgttttgag atcgaagaag attataaaaa tcgtcagttt ctggggcctg aaggaaatgt
     1201 ggatgttgag ttgattgata agagcacaaa cagatacagc gtttggttcc ccactgctgg
     1261 ctggtatctg tggtcagcca caggcctcgg cttcctggta agggatgagg tcacagtgac
     1321 gattgcgttt ggttcctgga gtcagcacct ggccctggac ctgcagcacc atgaacagtg
     1381 gctggtgggc ggccccttgt ttgatgtcac tgcagagcca gaggaggctg tcgccgaaat
     1441 ccacctcccc cacttcatct ccctccaagc aggtgaggtg gacgtctcct ggtttctcgt
     1501 tgcccatttt aagaatagag ggatggtcct ggagcatcca gcccgggtgg agcctttcta
     1561 tgctgtcctg gaaagcccca gcttcgccct gatgggcatc ctgctgcgga tcgccagtgg
     1621 gactcgcctc tccatcccca tcacttccaa cacattgatc tattatcacc cccaccccga
     1681 agatattaag ttccacttgt accttgtccc cagcgacgcc ttgctaacaa aggcgataga
     1741 tgatgaggaa gatcgcttcc atggtgtgcg cctgcagact tcgcccccaa tggaacccct
     1801 gaactttggt tccagttata ttgtgtctaa ttctgctaac ctgaaagtaa tgcccaagga
     1861 gttgaaattg tcctacagga gccctggaga aattcagcac ttctcaaaat tctatgctgg
     1921 gcagatgaag gaacccattc aacttgagat tactgaaaaa agacatggga ctttggtgtg
     1981 ggatactgag gtgaagccag tggatctcca gcttgtagct gcatcagccc ctcctccttt
     2041 ctcaggtgca gcctttgtga aggagaacca ccggcaactc caagccagga tgggggacct
     2101 gaaaggggtg ctcgatgatc tccaagacaa tgaggttctt actgagaatg agaaggagct
     2161 ggtggagcag gaaaagacac ggcagagcaa gaatgaggcc ttgctgagca tggtggagaa
     2221 gaaaggggac ctggccctgg acgtgctctt cagaagcatt agtgaaaggg acccttacct
     2281 cgtgtcctat cttagacagc agaatttgga cccagctttc ttgtacaaag tggttgatat
     2341 cgactacaag gacgatgacg acaagtagta ataacattgg aagtggataa tctagagggc
     2401 cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct
     2461 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2521 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2581 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2641 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2701 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2761 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2821 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2881 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2941 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3001 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3061 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3121 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     3181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3241 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3301 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3361 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3421 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3481 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     3541 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3601 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3661 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3721 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3781 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3841 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3901 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3961 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4021 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4081 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4141 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4201 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4261 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4321 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4381 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4441 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4501 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4561 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4621 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4681 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4741 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4801 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4861 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4921 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4981 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5041 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5101 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5161 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5221 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5281 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5341 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5401 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5461 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5521 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5581 tggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5641 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5701 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5761 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5821 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5881 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5941 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6001 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6061 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6121 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6181 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6241 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6301 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6361 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6421 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6481 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6541 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6601 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6661 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6721 cttccttttt cattattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6781 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6841 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     6901 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     6961 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     7021 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
     7081 acgcgtt
//
