LOCUS       sequence-332712-        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 (F405G).
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 F405G + S297A
FEATURES             Location/Qualifiers
     source          1..3381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     128..147
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     381..398
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(557..584)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(676..762)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     811..833
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     825..842
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     826..842
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    858..957
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(1795..1894)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1911..1930)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1912..1930)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1935..1951)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1935..1951)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2053..2072)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2064..2873
                     /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     complement(2131..2150)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(3020..3381,1..227)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
       61 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
      121 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
      181 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
      241 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
      301 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
      361 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
      421 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
      481 ccgactggaa agcgggcagt gagcgcaacg caattaatac gcgtaccgct agccaggaag
      541 agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct
      601 ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa
      661 atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa
      721 acgaaaggcc cagtcttccg actgagcctt tcgttttatt tgatgcctgg cagttcccta
      781 ctctcgcgtt aacgctagca tggatgtttt cccagtcacg acgttgtaaa acgacggcca
      841 gtcttaagct cgggccccaa ataatgattt tattttgact gatagtgacc tgttcgttgc
      901 aacaaattga tgagcaatgc ttttttataa tgccaacttt gtacaaaaaa gcaggcttcg
      961 aaggagatag aaccatgggg cctgaaggaa atgtggatgt tgagttgatt gataagagca
     1021 caaacagata cagcgtttgg ttccccactg ctggctggta tctgtggtca gccacaggcc
     1081 tcggcttcct ggtaagggat gaggtcacag tgacgattgc gtttggttcc tggagtcagc
     1141 acctggccct ggacctgcag caccatgaac agtggctggt gggcggcccc ttgtttgatg
     1201 tcactgcaga gccagaggag gctgtcgccg aaatccacct cccccacttc atctccctcc
     1261 aagcaggtga ggtggacgtc tcctggtttc tcgttgccca ttttaagaat gaagggatgg
     1321 tcctggagca tccagcccgg gtggagcctt tctatgctgt cctggaaagc cccagcttcg
     1381 ccctgatggg catcctgctg cggatcgcca gtgggactcg cctctccatc cccatcactt
     1441 ccaacacatt gatctattat cacccccacc ccgaagatat taagttccac ttgtaccttg
     1501 tccccagcga cgccttgcta acaaaggcga tagatgatga ggaagatcgc ttccatggtg
     1561 tgcgcctgca gacttcgccc ccaatggaac ccctgaactt tggttccagt tatattgtgt
     1621 ctaattctgc taacctgaaa gtaatgccca aggagttgaa attgtcctac aggagccctg
     1681 gagaaattca gcacttctca aaaggctatg ctgggcagat gaaggaaccc attcaacttg
     1741 agattactga aaaaagacat gggactttgg tgtgggatac tgaggtgaag ccagacccag
     1801 ctttcttgta caaagttggc attataagaa agcattgctt atcaatttgt tgcaacgaac
     1861 aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgatatc ccctatagtg
     1921 agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct
     1981 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
     2041 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat
     2101 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     2161 aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga
     2221 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     2281 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     2341 ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg
     2401 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     2461 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     2521 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     2581 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     2641 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     2701 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     2761 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     2821 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat
     2881 tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct
     2941 catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag
     3001 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3061 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3121 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     3181 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3241 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3301 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3361 ccagcttgga gcgaacgacc t
//
