LOCUS       sequence-332721-        7673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the dTAG-CARD8 fusion protein for 
            controllable activation with dTAG ligands (dTAG13, dTAGV-1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7673)
  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 7673)
  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: pLEX305-N-dTAG-CARD8
FEATURES             Location/Qualifiers
     source          1..7673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(25..221)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(33..243)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      39..174
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             complement(474..518)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    703..936
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    1429..1554
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             1601..1781
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(1782..2008)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(2036..2056)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2036..2054)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2075..2091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2088..2110)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2099..2115
                     /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(2123..2129,2130..2147,2148..2153))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2168..2189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2306..2323)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2477..3065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2557..2576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3212..4021)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3935..3954
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4013..4032
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     4134..4150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4155..4174
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4155..4173
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4191..4290
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(5911..5935)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(5951..5977)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(5981..6007)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             complement(5981..6007)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(join(6014..6334,6335..6337))
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     promoter        complement(6346..6846)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6409..6427)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    6904..7021
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(7064..7666)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGAS"
     primer_bind     complement(7150..7170)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     7647..7666
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 tccggtggta cctctagaaa gctttttgca aaagcctagg cctccaaaaa agcctcctca
       61 ctacttctgg aatagctcag aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat
      121 tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg
      181 agttaggggc gggactatgg ttgctgacta attgagacgc atgctttgca tacttctgcc
      241 tgctggggag cctggggact ttccacacct ggttgctgac taattgagat gcaatgcatg
      301 ggtcccctcg gggttgggag gtgggtctga aacgataatg gtgaatatcc ctgcctaact
      361 ctattcacta tagaaagtac agcaaaaact attcttaaac ctaccaagcc tcctactatc
      421 attatgaata attttatata ccacagccaa tttgttatgt taaaccaatt ccacaaactt
      481 gcccatttat ctaattccaa taattcttgt tcattctttt cttgctggtt ttgcgattct
      541 tcaattaagg agtgtattaa gcttgtgtaa ttgttaattt ctctgtccca ctccatccag
      601 gtcgtgtgat tccaaatctg ttccagagat ttattactcc aactagcatt ccaaggcaca
      661 gcagtggtgc aaatgagttt tccagagcaa ccccaaatcc ccaggagctg ttgatccttt
      721 aggtatcttt ccacagccag gattcttgcc tggagctgct tgatgcccca gactgtgagt
      781 tgcaacagat gctgttgcgc ctcaatagcc ctcagcaaat tgttctgctg ctgcactata
      841 ccagacaata attgtctggc ctgtaccgtc agcgtcattg acgctgcgcc catagtgctt
      901 cctgctgctc ccaagaaccc aaggaacaaa gctcctattc ccactgctct tttttctctc
      961 tgcaccactc ttctctttgc cttggtgggt gctactccta atggttcaat ttttactact
     1021 ttatatttat ataattcact tctccaattg tccctcatat ctcctcctcc aggtctgaag
     1081 atcagcggcc gcttgctgtg cggtggtctt acttttgttt tgctcttcct ctatcttgtc
     1141 taaagcttcc ttggtgtctt ttatctctat cctttgatgc acacaataga gggttgctac
     1201 tgtattatat aatgatctaa gttcttctga tcctgtctga agggatggtt gtagctgtcc
     1261 cagtatttgt ctacagcctt ctgatgtttc taacaggcca ggattaactg cgaatcgttc
     1321 tagctccctg cttgcccata ctatatgttt taatttatat tttttctttc cccctggcct
     1381 taaccgaatt ttttcccatc gcgatctaat tctcccccgc ttaatactga cgctctcgca
     1441 cccatctctc tccttctagc ctccgctagt caaaattttt ggcgtactca ccagtcgccg
     1501 cccctcgcct cttgccgtgc gcgcttcagc aagccgagtc ctgcgtcgag agagctcctc
     1561 tggtttccct ttcgctttca agtccctgtt cgggcgccac tgctagagat tttccacact
     1621 gactaaaagg gtctgaggga tctctagtta ccagagtcac acaacagacg ggcacacact
     1681 acttgaagca ctcaaggcaa gctttattga ggcttaagca gtgggttccc tagttagcca
     1741 gagagctccc aggctcagat ctggtctaac cagagagacc cgtttatgta tcgagctagg
     1801 cacttaaata caatatctct gcaatgcggc aattcagtgg ttcgtccaat ccatgtcaga
     1861 cccgtctgtt gccttcctaa taaggcacga tcgtaccacc ttacttccac caatcggcat
     1921 gcacggtgct ttttctctcc ttgtaaggca tgttgctaac tcatcgttac catgttgcaa
     1981 gactacaaga gtattgcata agactacatt aagcttgcag ctccagcttt tgttcccttt
     2041 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     2101 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2161 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     2221 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2281 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2341 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2401 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2461 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2521 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2581 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2641 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2701 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2761 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2821 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2881 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2941 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3001 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3061 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gacgcgtaac
     3121 tcacgttaag ggattttggt catgagcttg cgccgtcccg tcaagtcagc gtaatgctct
     3181 gccagtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa
     3241 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3301 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3361 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3421 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     3481 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3541 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3601 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3661 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgtttttc
     3721 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3781 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3841 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3901 agcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3961 aatcagcatc catgttggaa tttaatcgcg gcctcgacgt ttcccgttga atatggctca
     4021 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat
     4081 ttttatcttg tgcaatgtaa catcagagat tttgagacac gggccagagc tgccaggaaa
     4141 cagctatgac catgtaatac gactcactat aggggatatc agctggatgg caaataatga
     4201 ttttattttg actgatagtg acctgttcgt tgcaacaaat tgataagcaa tgctttctta
     4261 taatgccaac tttgtacaag aaagctgggt cttacaaatt ctgctgtcta agataggaca
     4321 cgaggtaagg gtccctttca ctaatgcttc tgaagagcac gtccagggcc aggtcccctt
     4381 tcttctccac catgctcagc aaggcctcat tcttgctctg ccgtgtcttt tcctgctcca
     4441 ccagctcctt ctcattctca gtaagaacct cattgtcttg gagatcatcg agcacccctt
     4501 tcaggtcccc catcctggct tggagttgcc ggtggttctc cttcacaaag gctgcacctg
     4561 agaaaggagg aggggctgat gcagctacaa gctggagatc cactggcttc acctcagtat
     4621 cccacaccaa agtcccatgt cttttttcag taatctcaag ttgaatgggt tccttcatct
     4681 gcccagcata gaattttgag aagtgctgaa tttctccagg gctcctgtag gacaatttca
     4741 actccttggg cattactttc aggttagcag aattagacac aatataactg gaaccaaagt
     4801 tcaggggttc cattgggggc gaagtctgca ggcgcacacc atggaagcga tcttcctcat
     4861 catctatcgc ctttgttagc aaggcgtcgc tggggacaag gtacaagtgg aacttaatat
     4921 cttcggggtg ggggtgataa tagatcaatg tgttggaagt gatggggatg gagaggcgag
     4981 tcccactggc gatccgcagc aggatgccca tcagagagaa gctggggctt tccaggacag
     5041 catagaaagg ctccacccgg gctggatgct ccaggaccat cccttcattc ttaaaatggg
     5101 caacgagaaa ccaggagacg tccacctcac ctgcttggag ggagatgaag tgggggaggt
     5161 ggatttcggc gacagcctcc tctggctctg cagtgacatc aaacaagggg ccgcccacca
     5221 gccactgttc atggtgctgc aggtccaggg ccaggtgctg actccaggaa ccaaacgcaa
     5281 tcgtcactgt gacctcatcc cttaccagga agccgaggcc tgtggctgac cacagatacc
     5341 agccagcagt ggggaaccaa acgctgtatc tgtttgtgct cttatcaatc aactcaacat
     5401 ccacatttcc ttcaggcccc agaaactgac gatttttata atcttcttcg atctcaaaac
     5461 agactttaga agcataagag gaaactattt gattctcttc tgagcaaatg tctcctgaat
     5521 cttgtccctc tgaagattcc tgctcttctg atacactggg aatgtccccc ccagatagtt
     5581 gacactcagg aacagcacgg aacaataatg gctctgcctc tgtctcatca tcttcttgga
     5641 aaaaatgtga gatgtcacaa agggtctcag aaacacaggg tagctccctg tataccgtat
     5701 catttgtcac acaggcctca gcctgaaaaa aaattccagt ttttgtgtat tgcagttccc
     5761 gtatgctatt gtcaaccaac agtttccgtg atccttgtag tctaatgagt ttggatgcat
     5821 ctatgttcct actggatcca ctgtcccgtc tcggcagctc ttcctcactg ctgctactct
     5881 tttctggaca ctcctttttt tccatggcga agcctgcttt tttgtacaaa cttgttgatc
     5941 tatcgatgcc tgcatagtcc gggacatcat acggatagcc ggcgtagtcg ggcacgtcgt
     6001 aggggtagcc gccttccagt tttagaagct ccacatcgaa gacgagagtg gcatgtggtg
     6061 ggatgatgcc tgggtgccca gtggcaccat aggcataatc tggagatata gtcagtttgg
     6121 ctctctgacc cacactcatc tgggcaaccc cttcttccca gcctcggatc acctcctgct
     6181 tgcctagcat aaacttaaag ggcttgtttc tgtcccggga ggaatcaact ttctttccat
     6241 cttcaagcat cccggtgtag tgcaccacgc aggtctggcc gcgcttgggg aaggtgcgcc
     6301 cgtctcctgg ggagatggtt tccacctgca ctcccatgct agcccctggg gagagaggtc
     6361 ggtgattcgg tcaacgaggg agccgactgc cgacgtgcgc tccggaggct tgcagaatgc
     6421 ggaacaccgc gcgggcagga acagggccca cactaccgcc ccacaccccg cctcccgcac
     6481 cgccccttcc cggccgctgc tctcggcgcg ccctgctgag cagccgctat tggccacagc
     6541 ccatcgcggt cggcgcgctg ccattgctcc ctggcgctgt ccgtctgcga gggtactagt
     6601 gagacgtgcg gcttccgttt gtcacgtccg gcacgccgcg aaccgcaagg aaccttcccg
     6661 acttaggggc ggagcaggaa gcgtcgccgg ggggcccaca agggtagcgg cgaagatccg
     6721 ggtgacgctg cgaacggacg tgaagaatgt gcgagaccca gggtcggcgc cgctgcgttt
     6781 cccggaacca cgcccagagc agccgcgtcc ctgcgcaaac ccagggctgc cttggaaaag
     6841 gcgcaacctg caggcctcga gccggcgcca aagtggatct ctgctgtccc tgtaataaac
     6901 ccgaaaattt tgaatttttg taatttgttt ttgtaattct ttagtttgta tgtctgttgc
     6961 tattatgtct actattcttt cccctgcact gtacccccca atcccccctt ttcttttaaa
     7021 attgtggatg aatactgcca tttgtctcaa gatctagaat tcgttaactg gcaccgggct
     7081 tgcgggtcat gcaccaggtg cgcggtcctt cgggcacctc gacgtcggcg gtgacggtga
     7141 agccgagccg ctcgtagaag gggaggttgc ggggcgcgga ggtctccagg aaggcgggca
     7201 ccccggcgcg ctcggccgcc tccactccgg ggagcacgac ggcgctgccc agacccttgc
     7261 cctggtggtc gggcgagacg ccgacggtgg ccaggaacca cgcgggctcc ttgggccggt
     7321 gcggcgccag gaggccttcc atctgttgct gcgcggccag ccgggaaccg ctcaactcgg
     7381 ccatgcgcgg gccgatctcg gcgaacaccg cccccgcttc gacgctctcc ggcgtggtcc
     7441 agaccgccac ggcggcgccg tcgtccgcga cccacacctt gccgatgtcg agcccgacgc
     7501 gcgtgaggaa gagttcttgc agctcggtga cccgctcgat gtggcggtcc ggatcgacgg
     7561 tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag ggtgcgtacg gccctgggga
     7621 cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc ggtcatggta agc
//
