LOCUS       sequence-332593-        6370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the CARD8 C-terminus with an interface III 
            mutation fused to ubiquitin (co-translationally processed) .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6370)
  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 6370)
  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 Ub-S297-FLAG L368G
FEATURES             Location/Qualifiers
     source          1..6370
                     /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    667..691
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             700..927
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     protein_bind    complement(1652..1676)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1684..1707
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1727..1744)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1733..1957
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2003..2431
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2090..2109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2300..2321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2440..2460)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2445..2774
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2625..2760
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2687..2706
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2841..3635
                     /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(2895..2914)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3505..3524
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3809..3930
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3846..3865)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3900..3919
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3979..3995)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3979..3995)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3992..4014)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4003..4019
                     /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(4027..4033,4034..4051,4052..4057))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4072..4093
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4210..4227)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4381..4969)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4461..4480)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5140..5931,5932..6000))
                     /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     5763..5782
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6001..6105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6180..6199)
                     /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 acccaagctg
      661 gctagcacaa gtttgtacaa aaaagcaggc ttcgccacca tgcagatctt cgtcaagacg
      721 ttaaccggta aaaccataac tctggaagtt gaaccatccg ataccatcga aaacgttaag
      781 gctaaaattc aagacaagga aggaattcca cctgatcaac aaagattgat ctttgccggt
      841 aagcagctcg aggacggtag aacgctgtct gattacaaca ttcagaagga gtcgacctta
      901 catcttgtct taagactaag aggtggttct ctgatgggca tcctgctgcg gatcgccagt
      961 gggactcgcc tctccatccc catcacttcc aacacattga tctattatca cccccacccc
     1021 gaagatatta agttccactt gtaccttgtc cccagcgacg ccttgctaac aaaggcgata
     1081 gatgatgagg aagatcgctt ccatggtgtg cgcctgcaga cttcgccccc aatggaaccc
     1141 ggcaactttg gttccagtta tattgtgtct aattctgcta acctgaaagt aatgcccaag
     1201 gagttgaaat tgtcctacag gagccctgga gaaattcagc acttctcaaa attctatgct
     1261 gggcagatga aggaacccat tcaacttgag attactgaaa aaagacatgg gactttggtg
     1321 tgggatactg aggtgaagcc agtggatctc cagcttgtag ctgcatcagc ccctcctcct
     1381 ttctcaggtg cagcctttgt gaaggagaac caccggcaac tccaagccag gatgggggac
     1441 ctgaaagggg tgctcgatga tctccaagac aatgaggttc ttactgagaa tgagaaggag
     1501 ctggtggagc aggaaaagac acggcagagc aagaatgagg ccttgctgag catggtggag
     1561 aagaaagggg acctggccct ggacgtgctc ttcagaagca ttagtgaaag ggacccttac
     1621 ctcgtgtcct atcttagaca gcagaatttg gacccagctt tcttgtacaa agtggttggg
     1681 cccgattaca aggatgacga cgataagtga taaacccgct gatcagcctc gactgtgcct
     1741 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     1801 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     1861 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     1921 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     1981 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2041 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2281 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2341 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2401 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     2461 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2521 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2581 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2641 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2701 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2761 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     2821 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     2881 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     2941 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3001 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3061 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3121 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3181 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3241 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     3301 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     3361 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     3421 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     3481 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     3541 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     3601 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     3661 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     3721 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     3781 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     3841 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3901 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     3961 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4021 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4081 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4141 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4201 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4261 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4321 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4381 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4441 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4501 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4561 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4621 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4681 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4741 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4801 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4861 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4921 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4981 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5041 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5101 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5161 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5221 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5281 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5341 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5401 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5461 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5521 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5581 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5641 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5701 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5761 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5821 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5881 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5941 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6001 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6061 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6121 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     6181 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     6241 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     6301 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     6361 tatacgcgtt
//
