LOCUS       sequence-335591-        7806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian purification construct for CARD8 with an 
            autoprocessing-deficient mutation.
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 S297A
FEATURES             Location/Qualifiers
     source          1..7806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     81..102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(221..241)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        226..555
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      406..541
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     468..487
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             622..1416
                     /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(676..695)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1286..1305
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1590..1711
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1627..1646)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1681..1700
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1760..1776)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1760..1776)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1773..1795)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1784..1800
                     /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(1808..1814,1815..1832,1833..1838))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1853..1874
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1991..2008)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2162..2750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2242..2261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2921..3712,3713..3781))
                     /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     3544..3563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3782..3886)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3961..3980)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4152..4531
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4532..4735
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4686..4706
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4780..4799
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4780..4798
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4846..4855
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6478..6498
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(6517..6519,6520..7230)
                     /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(6559..6580)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     7167..7188
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7231..7254
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(7276..7294)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7277..7294)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(7314..7331)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7320..7544
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(7590..7806,1..212)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7677..7696)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
       61 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
      121 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
      181 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
      241 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
      301 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
      361 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
      421 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
      481 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
      541 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
      601 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
      661 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
      721 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
      781 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
      841 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
      901 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
      961 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     1021 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     1081 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     1141 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     1201 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     1261 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     1321 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     1381 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     1441 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     1501 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     1561 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     1621 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     1681 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     1741 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1801 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     1861 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     1921 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     1981 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2041 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2101 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2161 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2221 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2281 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2341 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2401 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2461 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2521 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2581 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2641 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2701 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2761 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2821 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     2881 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     2941 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3001 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3061 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3121 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3181 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3241 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3301 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3361 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3421 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3481 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3541 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3601 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3661 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3721 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3781 tactcttcct ttttcattat tattgaagca tttatcaggg ttattgtctc atgagcggat
     3841 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     3901 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     3961 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     4021 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     4081 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     4141 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     4201 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     4261 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     4321 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     4381 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     4441 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     4501 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     4561 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     4621 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     4681 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     4741 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     4801 gacccaagct tggtaccgag ctcggatccc tacttccaat ccaatgccac catggaaaaa
     4861 aaggagtgtc cagaaaagag tagcagcagt gaggaagagc tgccgagacg ggacagtgga
     4921 tccagtagga acatagatgc atccaaactc attagactac aaggatcacg gaaactgttg
     4981 gttgacaata gcatacggga actgcaatac acaaaaactg gaattttttt tcaggctgag
     5041 gcctgtgtga caaatgatac ggtatacagg gagctaccct gtgtttctga gaccctttgt
     5101 gacatctcac attttttcca agaagatgat gagacagagg cagagccatt attgttccgt
     5161 gctgttcctg agtgtcaact atctgggggg gacattccca gtgtatcaga agagcaggaa
     5221 tcttcagagg gacaagattc aggagacatt tgctcagaag agaatcaaat agtttcctct
     5281 tatgcttcta aagtctgttt tgagatcgaa gaagattata aaaatcgtca gtttctgggg
     5341 cctgaaggaa atgtggatgt tgagttgatt gataagagca caaacagata cagcgtttgg
     5401 ttccccactg ctggctggta tctgtggtca gccacaggcc tcggcttcct ggtaagggat
     5461 gaggtcacag tgacgattgc gtttggttcc tggagtcagc acctggccct ggacctgcag
     5521 caccatgaac agtggctggt gggcggcccc ttgtttgatg tcactgcaga gccagaggag
     5581 gctgtcgccg aaatccacct cccccacttc atctccctcc aagcaggtga ggtggacgtc
     5641 tcctggtttc tcgttgccca ttttaagaat gaagggatgg tcctggagca tccagcccgg
     5701 gtggagcctt tctatgctgt cctggaaagc cccagcttcg ccctgatggg catcctgctg
     5761 cggatcgcca gtgggactcg cctctccatc cccatcactt ccaacacatt gatctattat
     5821 cacccccacc ccgaagatat taagttccac ttgtaccttg tccccagcga cgccttgcta
     5881 acaaaggcga tagatgatga ggaagatcgc ttccatggtg tgcgcctgca gacttcgccc
     5941 ccaatggaac ccctgaactt tggttccagt tatattgtgt ctaattctgc taacctgaaa
     6001 gtaatgccca aggagttgaa attgtcctac aggagccctg gagaaattca gcacttctca
     6061 aaattctatg ctgggcagat gaaggaaccc attcaacttg agattactga aaaaagacat
     6121 gggactttgg tgtgggatac tgaggtgaag ccagtggatc tccagcttgt agctgcatca
     6181 gcccctcctc ctttctcagg tgcagccttt gtgaaggaga accaccggca actccaagcc
     6241 aggatggggg acctgaaagg ggtgctcgat gatctccaag acaatgaggt tcttactgag
     6301 aatgagaagg agctggtgga gcaggaaaag acacggcaga gcaagaatga ggccttgctg
     6361 agcatggtgg agaagaaagg ggacctggcc ctggacgtgc tcttcagaag cattagtgaa
     6421 agggaccctt acctcgtgtc ctatcttaga cagcagaatg gcattggtag tgggagcgaa
     6481 aacctttact tccagggcaa cggcagcagc ggatccgtga gcaagggcga ggagctgttc
     6541 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     6601 gtgcgcggcg agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc
     6661 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     6721 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     6781 cccgaaggct acgtccagga gcgcaccatc tccttcaagg acgacggcac ctacaagacc
     6841 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     6901 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac
     6961 aacgtctata tcacggccga caagcagaag aacggcatca aggcgaactt caagatccgc
     7021 cacaacgtcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     7081 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc
     7141 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     7201 atcactctcg gcatggacga gctgtacaag gactacaagg acgatgacga caagtagtaa
     7261 tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     7321 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     7381 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     7441 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     7501 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     7561 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     7621 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     7681 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     7741 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     7801 tgatta
//
