LOCUS       sequence-332702-        3381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR221 for Gateway shuttling. Contains the CARD8 
            autoprocessing-deficient FIIND (L162M-P434, no stop codon) that 
            cannot bind DPP9 at site I (E274R).
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 E274R + S297A
FEATURES             Location/Qualifiers
     source          1..3381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(199..298)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(314..331)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(314..330)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(323..345)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      394..480
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      572..599
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(758..775)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(929..1517)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1009..1028)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1664..2473)
                     /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     2387..2406
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2465..2484
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2586..2602
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2586..2602
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2607..2626
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2607..2625
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2643..2742
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 ggtccagggc caggtgctga ctccaggaac caaacgcaat cgtcactgtg acctcatccc
       61 ttaccaggaa gccgaggcct gtggctgacc acagatacca gccagcagtg gggaaccaaa
      121 cgctgtatct gtttgtgctc ttatcaatca actcaacatc cacatttcct tcaggcccca
      181 tggttctatc tccttcgaag cctgcttttt tgtacaaagt tggcattata aaaaagcatt
      241 gctcatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttggg
      301 gcccgagctt aagactggcc gtcgttttac aacgtcgtga ctgggaaaac atccatgcta
      361 gcgttaacgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcggaa
      421 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
      481 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc
      541 ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga tggccttttt
      601 gcgtttctac aaactcttcc tggctagcgg tacgcgtatt aattgcgttg cgctcactgc
      661 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
      721 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
      781 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
      841 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
      901 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
      961 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1021 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1081 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1141 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1201 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     1261 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     1321 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     1381 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     1441 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     1501 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     1561 acgacgcgta actcacgtta agggattttg gtcatgagct tgcgccgtcc cgtcaagtca
     1621 gcgtaatgct ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga
     1681 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     1741 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     1801 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     1861 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     1921 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     1981 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     2041 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     2101 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     2161 atgctgtttt tccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     2221 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     2281 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     2341 gcttcccata caagcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     2401 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgac gtttcccgtt
     2461 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     2521 atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acgggccaga
     2581 gctgccagga aacagctatg accatgtaat acgactcact ataggggata tcagctggat
     2641 ggcaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa attgataagc
     2701 aatgctttct tataatgcca actttgtaca agaaagctgg gtctggcttc acctcagtat
     2761 cccacaccaa agtcccatgt cttttttcag taatctcaag ttgaatgggt tccttcatct
     2821 gcccagcata gaattttgag aagtgctgaa tttctccagg gctcctgtag gacaatttca
     2881 actccttggg cattactttc aggttagcag aattagacac aatataactg gaaccaaagt
     2941 tcaggggttc cattgggggc gaagtctgca ggcgcacacc atggaagcga tcttcctcat
     3001 catctatcgc ctttgttagc aaggcgtcgc tggggacaag gtacaagtgg aacttaatat
     3061 cttcggggtg ggggtgataa tagatcaatg tgttggaagt gatggggatg gagaggcgag
     3121 tcccactggc gatccgcagc aggatgccca tcagggcgaa gctggggctt tccaggacag
     3181 catagaaagg ctccacccgg gctggatgct ccaggaccat ccctctattc ttaaaatggg
     3241 caacgagaaa ccaggagacg tccacctcac ctgcttggag ggagatgaag tgggggaggt
     3301 ggatttcggc gacagcctcc tctggctctg cagtgacatc aaacaagggg ccgcccacca
     3361 gccactgttc atggtgctgc a
//
