LOCUS       sequence-342403-        7087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of CARD8-FLAG with a site III mutation (F405G).
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 F405G
FEATURES             Location/Qualifiers
     source          1..7087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(65..85)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        70..399
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      250..385
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     312..331
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             466..1260
                     /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(520..539)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1130..1149
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1434..1555
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1471..1490)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1525..1544
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1604..1620)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1604..1620)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1617..1639)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1628..1644
                     /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(1652..1658,1659..1676,1677..1682))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1697..1718
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1835..1852)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2006..2594)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2086..2105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2765..3556,3557..3625))
                     /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     3388..3407
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3626..3730)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3805..3824)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3996..4375
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4376..4579
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4530..4550
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4624..4643
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4624..4642
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(6305..6329)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6337..6360
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(6401..6419)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6402..6419)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(6439..6456)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6445..6669
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6715..7087,1..56)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6802..6821)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7012..7033
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
       61 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
      121 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
      181 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
      241 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
      301 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
      361 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
      421 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
      481 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
      541 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
      601 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
      661 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
      721 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
      781 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
      841 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
      901 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
      961 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     1021 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     1081 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     1141 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     1201 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     1261 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     1321 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     1381 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     1441 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     1501 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     1561 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     1621 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     1681 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     1741 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1801 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1861 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1921 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1981 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2041 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2101 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2161 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2221 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2281 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2341 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2401 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2461 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2521 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2581 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2641 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2701 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2761 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2821 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2881 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     2941 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3001 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3061 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3121 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3181 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3241 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3301 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3361 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3421 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3481 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3541 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3601 cgacacggaa atgttgaata ctcatactct tcctttttca ttattattga agcatttatc
     3661 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3721 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     3781 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3841 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     3901 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     3961 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     4021 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     4081 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     4141 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     4201 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     4261 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     4321 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     4381 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     4441 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     4501 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     4561 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
     4621 aattaatacg actcactata gggagaccca agcttggtac cgagctcgga tccctacttc
     4681 caatccaatg caatggaaaa aaaggagtgt ccagaaaaga gtagcagcag tgaggaagag
     4741 ctgccgagac gggacagtgg atccagtagg aacatagatg catccaaact cattagacta
     4801 caaggatcac ggaaactgtt ggttgacaat agcatacggg aactgcaata cacaaaaact
     4861 ggaatttttt ttcaggctga ggcctgtgtg acaaatgata cggtatacag ggagctaccc
     4921 tgtgtttctg agaccctttg tgacatctca cattttttcc aagaagatga tgagacagag
     4981 gcagagccat tattgttccg tgctgttcct gagtgtcaac tatctggggg ggacattccc
     5041 agtgtatcag aagagcagga atcttcagag ggacaagatt caggagacat ttgctcagaa
     5101 gagaatcaaa tagtttcctc ttatgcttct aaagtctgtt ttgagatcga agaagattat
     5161 aaaaatcgtc agtttctggg gcctgaagga aatgtggatg ttgagttgat tgataagagc
     5221 acaaacagat acagcgtttg gttccccact gctggctggt atctgtggtc agccacaggc
     5281 ctcggcttcc tggtaaggga tgaggtcaca gtgacgattg cgtttggttc ctggagtcag
     5341 cacctggccc tggacctgca gcaccatgaa cagtggctgg tgggcggccc cttgtttgat
     5401 gtcactgcag agccagagga ggctgtcgcc gaaatccacc tcccccactt catctccctc
     5461 caagcaggtg aggtggacgt ctcctggttt ctcgttgccc attttaagaa tgaagggatg
     5521 gtcctggagc atccagcccg ggtggagcct ttctatgctg tcctggaaag ccccagcttc
     5581 tctctgatgg gcatcctgct gcggatcgcc agtgggactc gcctctccat ccccatcact
     5641 tccaacacat tgatctatta tcacccccac cccgaagata ttaagttcca cttgtacctt
     5701 gtccccagcg acgccttgct aacaaaggcg atagatgatg aggaagatcg cttccatggt
     5761 gtgcgcctgc agacttcgcc cccaatggaa cccctgaact ttggttccag ttatattgtg
     5821 tctaattctg ctaacctgaa agtaatgccc aaggagttga aattgtccta caggagccct
     5881 ggagaaattc agcacttctc aaaaggctat gctgggcaga tgaaggaacc cattcaactt
     5941 gagattactg aaaaaagaca tgggactttg gtgtgggata ctgaggtgaa gccagtggat
     6001 ctccagcttg tagctgcatc agcccctcct cctttctcag gtgcagcctt tgtgaaggag
     6061 aaccaccggc aactccaagc caggatgggg gacctgaaag gggtgctcga tgatctccaa
     6121 gacaatgagg ttcttactga gaatgagaag gagctggtgg agcaggaaaa gacacggcag
     6181 agcaagaatg aggccttgct gagcatggtg gagaagaaag gggacctggc cctggacgtg
     6241 ctcttcagaa gcattagtga aagggaccct tacctcgtgt cctatcttag acagcagaat
     6301 ttggacccag ctttcttgta caaagtggtt gatatcgact acaaggacga tgacgacaag
     6361 tagtaataac attggaagtg gataatctag agggccctat tctatagtgt cacctaaatg
     6421 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     6481 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     6541 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     6601 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     6661 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     6721 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     6781 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     6841 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     6901 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     6961 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     7021 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     7081 ttttgcc
//
