LOCUS       sequence-332705-        4161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR221 for Gateway shuttling. Contains CARD8 with a site III 
            mutation (R394E) with sgRNA binding site mutations .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4161)
  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 4161)
  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 PAM R394E
FEATURES             Location/Qualifiers
     source          1..4161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(562..589)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(681..767)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     816..838
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     830..847
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     831..847
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    863..962
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(2580..2679)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2696..2715)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2697..2715)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2720..2736)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2720..2736)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2838..2857)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2849..3658
                     /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(2916..2935)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(3805..4161,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta ccgctagcca
      541 ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga
      601 tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg
      661 ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag
      721 ataaaacgaa aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt
      781 ccctactctc gcgttaacgc tagcatggat gttttcccag tcacgacgtt gtaaaacgac
      841 ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag tgacctgttc
      901 gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtaca aaaaagcagg
      961 cttcgccatg gaaaaaaagg agtgtccaga aaagagtagc agcagtgagg aagagctgcc
     1021 gagacgggac agtggatcca gtaggaacat agatgcatcc aaactcatta gactacaagg
     1081 atcacggaaa ctgttggttg acaatagcat acgggaactg caatacacaa aaactggaat
     1141 tttttttcag gctgaggcct gtgtgacaaa tgatacggta tacagggagc taccctgtgt
     1201 ttctgagacc ctttgtgaca tctcacattt tttccaagaa gatgatgaga cagaggcaga
     1261 gccattattg ttccgtgctg ttcctgagtg tcaactatct gggggggaca ttcccagtgt
     1321 atcagaagag caggaatctt cagagggaca agattcagga gacatttgct cagaagagaa
     1381 tcaaatagtt tcctcttatg cttctaaagt ctgttttgag atcgaagaag attataaaaa
     1441 tcgtcagttt ctggggcctg aaggaaatgt ggatgttgag ttgattgata agagcacaaa
     1501 cagatacagc gtttggttcc ccactgctgg ctggtatctg tggtcagcca caggcctcgg
     1561 cttcctggta agggatgagg tcacagttac catcgccttc ggcagctgga gtcagcacct
     1621 ggccctggac ctgcagcacc atgaacagtg gctggtgggc ggccccttgt ttgatgtcac
     1681 tgcagagcca gaggaggctg tcgccgaaat ccacctcccc cacttcatct ccctccaagc
     1741 aggtgaggtg gacgtctcct ggtttctcgt tgcccatttt aagaatgaag ggatggtcct
     1801 ggagcatcca gcccgggtgg agcctttcta tgctgtcctg gaaagcccca gcttctctct
     1861 gatgggcatc ctgctgcgga tcgccagtgg gactcgcctc tccatcccca tcacttccaa
     1921 cacattgatc tattatcacc cccaccccga agatattaag ttccacttgt accttgtccc
     1981 cagcgacgcc ttgctaacaa aggcgataga tgatgaggaa gatcgcttcc atggtgtgcg
     2041 cctgcagact tcgcccccaa tggaacccct gaactttggt tccagttata ttgtgtctaa
     2101 ttctgctaac ctgaaagtaa tgcccaagga gttgaaattg tcctacgaga gccctggaga
     2161 aattcagcac ttctcaaaat tctatgctgg gcagatgaag gaacccattc aacttgagat
     2221 tactgaaaaa agacatggga ctttggtgtg ggatactgag gtgaagccag tggatctcca
     2281 gcttgtagct gcatcagccc ctcctccttt ctcaggtgca gcctttgtga aggagaacca
     2341 ccggcaactc caagccagga tgggggacct gaaaggggtg ctcgatgatc tccaagacaa
     2401 tgaggttctt actgagaatg agaaggagct ggtggagcag gaaaagacac ggcagagcaa
     2461 gaatgaggcc ttgctgagca tggtggagaa gaaaggggac ctggccctgg acgtgctctt
     2521 cagaagcatt agtgaaaggg acccttacct cgtgtcctat cttagacagc agaatttgga
     2581 cccagctttc ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa
     2641 cgaacaggtc actatcagtc aaaataaaat cattatttgc catccagctg atatccccta
     2701 tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa
     2761 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
     2821 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc
     2881 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     2941 cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt
     3001 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     3061 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     3121 tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata
     3181 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     3241 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     3301 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     3361 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     3421 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     3481 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     3541 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     3601 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     3661 agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc
     3721 aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc
     3781 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3841 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3901 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3961 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4021 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4081 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4141 acagcccagc ttggagcgaa c
//
