LOCUS       sequence-332694-        3381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR221 for Gateway shuttling. Contains the 
            autoprocessing-deficient CARD8 FIIND (L162M-P434, no stop codon) 
            with an interface III mutation (R394E).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3381)
  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 3381)
  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: pDONR221 CARD8 FIIND R394E + S297A
FEATURES             Location/Qualifiers
     source          1..3381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(198..297)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(313..330)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(313..329)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(322..344)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      393..479
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      571..598
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(757..774)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(928..1516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1008..1027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1663..2472)
                     /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     2386..2405
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2464..2483
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2585..2601
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2585..2601
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2606..2625
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2606..2624
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2642..2741
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gtccagggcc aggtgctgac tccaggaacc aaacgcaatc gtcactgtga cctcatccct
       61 taccaggaag ccgaggcctg tggctgacca cagataccag ccagcagtgg ggaaccaaac
      121 gctgtatctg tttgtgctct tatcaatcaa ctcaacatcc acatttcctt caggccccat
      181 ggttctatct ccttcgaagc ctgctttttt gtacaaagtt ggcattataa aaaagcattg
      241 ctcatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgggg
      301 cccgagctta agactggccg tcgttttaca acgtcgtgac tgggaaaaca tccatgctag
      361 cgttaacgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcggaag
      421 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
      481 cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg gcaggacgcc
      541 cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat ggcctttttg
      601 cgtttctaca aactcttcct ggctagcggt acgcgtatta attgcgttgc gctcactgcc
      661 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
      721 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
      781 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
      841 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
      901 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
      961 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     1021 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     1081 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     1141 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     1201 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     1261 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     1321 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     1381 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     1441 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     1501 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     1561 cgacgcgtaa ctcacgttaa gggattttgg tcatgagctt gcgccgtccc gtcaagtcag
     1621 cgtaatgctc tgccagtgtt acaaccaatt aaccaattct gattagaaaa actcatcgag
     1681 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     1741 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     1801 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     1861 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     1921 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     1981 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     2041 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     2101 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     2161 tgctgttttt ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     2221 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     2281 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     2341 cttcccatac aagcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     2401 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgacg tttcccgttg
     2461 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     2521 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca cgggccagag
     2581 ctgccaggaa acagctatga ccatgtaata cgactcacta taggggatat cagctggatg
     2641 gcaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca
     2701 atgctttctt ataatgccaa ctttgtacaa gaaagctggg tctggcttca cctcagtatc
     2761 ccacaccaaa gtcccatgtc ttttttcagt aatctcaagt tgaatgggtt ccttcatctg
     2821 cccagcatag aattttgaga agtgctgaat ttctccaggg ctctcgtagg acaatttcaa
     2881 ctccttgggc attactttca ggttagcaga attagacaca atataactgg aaccaaagtt
     2941 caggggttcc attgggggcg aagtctgcag gcgcacacca tggaagcgat cttcctcatc
     3001 atctatcgcc tttgttagca aggcgtcgct ggggacaagg tacaagtgga acttaatatc
     3061 ttcggggtgg gggtgataat agatcaatgt gttggaagtg atggggatgg agaggcgagt
     3121 cccactggcg atccgcagca ggatgcccat cagggcgaag ctggggcttt ccaggacagc
     3181 atagaaaggc tccacccggg ctggatgctc caggaccatc ccttcattct taaaatgggc
     3241 aacgagaaac caggagacgt ccacctcacc tgcttggagg gagatgaagt gggggaggtg
     3301 gatttcggcg acagcctcct ctggctctgc agtgacatca aacaaggggc cgcccaccag
     3361 ccactgttca tggtgctgca g
//
