LOCUS       sequence-332590-        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 F405G
FEATURES             Location/Qualifiers
     source          1..6370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    287..311
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             320..547
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     protein_bind    complement(1272..1296)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1304..1327
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1347..1364)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1353..1577
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1623..2051
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1710..1729)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1920..1941
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2060..2080)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2065..2394
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2245..2380
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2307..2326
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2461..3255
                     /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(2515..2534)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3125..3144
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3429..3550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3466..3485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3520..3539
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3599..3615)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3599..3615)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3612..3634)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3623..3639
                     /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(3647..3653,3654..3671,3672..3677))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3692..3713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3830..3847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4001..4589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4081..4100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4760..5551,5552..5620))
                     /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     5383..5402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5621..5725)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5800..5819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5991..6370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagcacaa gtttgtacaa
      301 aaaagcaggc ttcgccacca tgcagatctt cgtcaagacg ttaaccggta aaaccataac
      361 tctggaagtt gaaccatccg ataccatcga aaacgttaag gctaaaattc aagacaagga
      421 aggaattcca cctgatcaac aaagattgat ctttgccggt aagcagctcg aggacggtag
      481 aacgctgtct gattacaaca ttcagaagga gtcgacctta catcttgtct taagactaag
      541 aggtggttct ctgatgggca tcctgctgcg gatcgccagt gggactcgcc tctccatccc
      601 catcacttcc aacacattga tctattatca cccccacccc gaagatatta agttccactt
      661 gtaccttgtc cccagcgacg ccttgctaac aaaggcgata gatgatgagg aagatcgctt
      721 ccatggtgtg cgcctgcaga cttcgccccc aatggaaccc ctgaactttg gttccagtta
      781 tattgtgtct aattctgcta acctgaaagt aatgcccaag gagttgaaat tgtcctacag
      841 gagccctgga gaaattcagc acttctcaaa aggctatgct gggcagatga aggaacccat
      901 tcaacttgag attactgaaa aaagacatgg gactttggtg tgggatactg aggtgaagcc
      961 agtggatctc cagcttgtag ctgcatcagc ccctcctcct ttctcaggtg cagcctttgt
     1021 gaaggagaac caccggcaac tccaagccag gatgggggac ctgaaagggg tgctcgatga
     1081 tctccaagac aatgaggttc ttactgagaa tgagaaggag ctggtggagc aggaaaagac
     1141 acggcagagc aagaatgagg ccttgctgag catggtggag aagaaagggg acctggccct
     1201 ggacgtgctc ttcagaagca ttagtgaaag ggacccttac ctcgtgtcct atcttagaca
     1261 gcagaatttg gacccagctt tcttgtacaa agtggttggg cccgattaca aggatgacga
     1321 cgataagtga taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     1381 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1441 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     1501 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     1561 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     1621 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1681 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1741 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1801 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1861 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1921 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1981 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2041 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2101 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2161 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2221 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2281 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     2341 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     2401 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     2461 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     2521 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     2581 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     2641 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     2701 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     2761 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     2821 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     2881 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     2941 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3001 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3061 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3121 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3181 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3241 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     3301 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3361 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3421 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3481 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3541 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     3601 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3661 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3721 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3781 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3841 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3901 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3961 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4021 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4081 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4141 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4201 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4261 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4321 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4381 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4441 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4501 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4561 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4621 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4681 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4741 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4801 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4861 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4921 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4981 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5041 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5101 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5161 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5221 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5281 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5341 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5401 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5461 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5521 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5581 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5641 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5701 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     5761 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     5821 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     5881 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     5941 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     6001 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     6061 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     6121 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     6181 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     6241 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     6301 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     6361 tattaccatg
//
