LOCUS       sequence-335654-        7806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian purification construct for CARD8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7806)
  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 7806)
  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 CARD8-TEV-GFP-FLAG
FEATURES             Location/Qualifiers
     source          1..7806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(75..95)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        80..409
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      260..395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     322..341
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             476..1270
                     /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(530..549)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1140..1159
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1444..1565
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1481..1500)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1535..1554
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1614..1630)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1614..1630)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1627..1649)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1638..1654
                     /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(1662..1668,1669..1686,1687..1692))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1707..1728
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1845..1862)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2016..2604)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2096..2115)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2775..3566,3567..3635))
                     /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     3398..3417
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3636..3740)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3815..3834)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4006..4385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4386..4589
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4540..4560
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4634..4653
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4634..4652
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4700..4709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6332..6352
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(6371..6373,6374..7084)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6413..6434)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     7021..7042
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7085..7108
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(7130..7148)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7131..7148)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(7168..7185)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7174..7398
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(7444..7806,1..66)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7531..7550)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7741..7762
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
       61 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
      121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
      181 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
      241 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
      301 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
      361 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
      421 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
      481 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
      541 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
      601 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
      661 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
      721 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
      781 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
      841 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
      901 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
      961 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     1021 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     1081 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     1141 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     1201 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     1261 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     1321 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     1381 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     1441 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1501 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     1561 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     1621 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1681 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1741 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1801 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1861 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1921 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1981 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2041 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2101 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2161 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2221 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2281 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2341 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2401 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2461 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2521 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2581 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2641 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2701 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2761 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2821 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2881 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     2941 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3001 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3061 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3121 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3181 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3241 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3301 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3361 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3421 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3481 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3541 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3601 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca ttattattga
     3661 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3721 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     3781 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3841 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     3901 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     3961 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     4021 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     4081 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     4141 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     4201 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     4261 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     4321 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     4381 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     4441 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     4501 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     4561 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
     4621 ggcttatcga aattaatacg actcactata gggagaccca agcttggtac cgagctcgga
     4681 tccctacttc caatccaatg ccaccatgga aaaaaaggag tgtccagaaa agagtagcag
     4741 cagtgaggaa gagctgccga gacgggacag tggatccagt aggaacatag atgcatccaa
     4801 actcattaga ctacaaggat cacggaaact gttggttgac aatagcatac gggaactgca
     4861 atacacaaaa actggaattt tttttcaggc tgaggcctgt gtgacaaatg atacggtata
     4921 cagggagcta ccctgtgttt ctgagaccct ttgtgacatc tcacattttt tccaagaaga
     4981 tgatgagaca gaggcagagc cattattgtt ccgtgctgtt cctgagtgtc aactatctgg
     5041 gggggacatt cccagtgtat cagaagagca ggaatcttca gagggacaag attcaggaga
     5101 catttgctca gaagagaatc aaatagtttc ctcttatgct tctaaagtct gttttgagat
     5161 cgaagaagat tataaaaatc gtcagtttct ggggcctgaa ggaaatgtgg atgttgagtt
     5221 gattgataag agcacaaaca gatacagcgt ttggttcccc actgctggct ggtatctgtg
     5281 gtcagccaca ggcctcggct tcctggtaag ggatgaggtc acagtgacga ttgcgtttgg
     5341 ttcctggagt cagcacctgg ccctggacct gcagcaccat gaacagtggc tggtgggcgg
     5401 ccccttgttt gatgtcactg cagagccaga ggaggctgtc gccgaaatcc acctccccca
     5461 cttcatctcc ctccaagcag gtgaggtgga cgtctcctgg tttctcgttg cccattttaa
     5521 gaatgaaggg atggtcctgg agcatccagc ccgggtggag cctttctatg ctgtcctgga
     5581 aagccccagc ttctctctga tgggcatcct gctgcggatc gccagtggga ctcgcctctc
     5641 catccccatc acttccaaca cattgatcta ttatcacccc caccccgaag atattaagtt
     5701 ccacttgtac cttgtcccca gcgacgcctt gctaacaaag gcgatagatg atgaggaaga
     5761 tcgcttccat ggtgtgcgcc tgcagacttc gcccccaatg gaacccctga actttggttc
     5821 cagttatatt gtgtctaatt ctgctaacct gaaagtaatg cccaaggagt tgaaattgtc
     5881 ctacaggagc cctggagaaa ttcagcactt ctcaaaattc tatgctgggc agatgaagga
     5941 acccattcaa cttgagatta ctgaaaaaag acatgggact ttggtgtggg atactgaggt
     6001 gaagccagtg gatctccagc ttgtagctgc atcagcccct cctcctttct caggtgcagc
     6061 ctttgtgaag gagaaccacc ggcaactcca agccaggatg ggggacctga aaggggtgct
     6121 cgatgatctc caagacaatg aggttcttac tgagaatgag aaggagctgg tggagcagga
     6181 aaagacacgg cagagcaaga atgaggcctt gctgagcatg gtggagaaga aaggggacct
     6241 ggccctggac gtgctcttca gaagcattag tgaaagggac ccttacctcg tgtcctatct
     6301 tagacagcag aatggcattg gtagtgggag cgaaaacctt tacttccagg gcaacggcag
     6361 cagcggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     6421 gctggacggc gacgtaaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc
     6481 caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     6541 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     6601 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     6661 catctccttc aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga
     6721 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     6781 ggggcacaag ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca
     6841 gaagaacggc atcaaggcga acttcaagat ccgccacaac gtcgaggacg gcagcgtgca
     6901 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     6961 caaccactac ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca
     7021 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     7081 caaggactac aaggacgatg acgacaagta gtaatctaga gggccctatt ctatagtgtc
     7141 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     7201 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     7261 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     7321 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     7381 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     7441 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     7501 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     7561 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     7621 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     7681 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     7741 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     7801 tataag
//
