LOCUS       sequence-332696-        4161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR221 for Gateway shuttling. Contains CARD8 with a site III 
            mutation (F370G) 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 F370G
FEATURES             Location/Qualifiers
     source          1..4161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..47)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(201..789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(281..300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(936..1745)
                     /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     1659..1678
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1737..1756
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1858..1874
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1858..1874
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1879..1898
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1879..1897
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1915..2014
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(3632..3731)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3747..3764)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3747..3763)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3756..3778)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      3827..3913
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4005..4032
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
       61 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      121 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
      181 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      241 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
      301 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
      361 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
      421 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
      481 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
      541 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
      601 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
      661 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
      721 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
      781 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgacgcg
      841 taactcacgt taagggattt tggtcatgag cttgcgccgt cccgtcaagt cagcgtaatg
      901 ctctgccagt gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
      961 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     1021 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     1081 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     1141 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     1201 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     1261 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     1321 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     1381 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     1441 tttccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     1501 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     1561 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     1621 tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     1681 tataaatcag catccatgtt ggaatttaat cgcggcctcg acgtttcccg ttgaatatgg
     1741 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat
     1801 atatttttat cttgtgcaat gtaacatcag agattttgag acacgggcca gagctgccag
     1861 gaaacagcta tgaccatgta atacgactca ctatagggga tatcagctgg atggcaaata
     1921 atgattttat tttgactgat agtgacctgt tcgttgcaac aaattgataa gcaatgcttt
     1981 cttataatgc caactttgta caagaaagct gggtccaaat tctgctgtct aagataggac
     2041 acgaggtaag ggtccctttc actaatgctt ctgaagagca cgtccagggc caggtcccct
     2101 ttcttctcca ccatgctcag caaggcctca ttcttgctct gccgtgtctt ttcctgctcc
     2161 accagctcct tctcattctc agtaagaacc tcattgtctt ggagatcatc gagcacccct
     2221 ttcaggtccc ccatcctggc ttggagttgc cggtggttct ccttcacaaa ggctgcacct
     2281 gagaaaggag gaggggctga tgcagctaca agctggagat ccactggctt cacctcagta
     2341 tcccacacca aagtcccatg tcttttttca gtaatctcaa gttgaatggg ttccttcatc
     2401 tgcccagcat agaattttga gaagtgctga atttctccag ggctcctgta ggacaatttc
     2461 aactccttgg gcattacttt caggttagca gaattagaca caatataact ggaaccaccg
     2521 ttcaggggtt ccattggggg cgaagtctgc aggcgcacac catggaagcg atcttcctca
     2581 tcatctatcg cctttgttag caaggcgtcg ctggggacaa ggtacaagtg gaacttaata
     2641 tcttcggggt gggggtgata atagatcaat gtgttggaag tgatggggat ggagaggcga
     2701 gtcccactgg cgatccgcag caggatgccc atcagagaga agctggggct ttccaggaca
     2761 gcatagaaag gctccacccg ggctggatgc tccaggacca tcccttcatt cttaaaatgg
     2821 gcaacgagaa accaggagac gtccacctca cctgcttgga gggagatgaa gtgggggagg
     2881 tggatttcgg cgacagcctc ctctggctct gcagtgacat caaacaaggg gccgcccacc
     2941 agccactgtt catggtgctg caggtccagg gccaggtgct gactccagct gccgaaggcg
     3001 atggtaactg tgacctcatc ccttaccagg aagccgaggc ctgtggctga ccacagatac
     3061 cagccagcag tggggaacca aacgctgtat ctgtttgtgc tcttatcaat caactcaaca
     3121 tccacatttc cttcaggccc cagaaactga cgatttttat aatcttcttc gatctcaaaa
     3181 cagactttag aagcataaga ggaaactatt tgattctctt ctgagcaaat gtctcctgaa
     3241 tcttgtccct ctgaagattc ctgctcttct gatacactgg gaatgtcccc cccagatagt
     3301 tgacactcag gaacagcacg gaacaataat ggctctgcct ctgtctcatc atcttcttgg
     3361 aaaaaatgtg agatgtcaca aagggtctca gaaacacagg gtagctccct gtataccgta
     3421 tcatttgtca cacaggcctc agcctgaaaa aaaattccag tttttgtgta ttgcagttcc
     3481 cgtatgctat tgtcaaccaa cagtttccgt gatccttgta gtctaatgag tttggatgca
     3541 tctatgttcc tactggatcc actgtcccgt ctcggcagct cttcctcact gctgctactc
     3601 ttttctggac actccttttt ttccatggcg aagcctgctt ttttgtacaa agttggcatt
     3661 ataaaaaagc attgctcatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa
     3721 atcattattt ggggcccgag cttaagactg gccgtcgttt tacaacgtcg tgactgggaa
     3781 aacatccatg ctagcgttaa cgcgagagta gggaactgcc aggcatcaaa taaaacgaaa
     3841 ggctcagtcg gaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     3901 gagtaggaca aatccgccgg gagcggattt gaacgttgtg aagcaacggc ccggagggtg
     3961 gcgggcagga cgcccgccat aaactgccag gcatcaaact aagcagaagg ccatcctgac
     4021 ggatggcctt tttgcgtttc tacaaactct tcctggctag cggtacgcgt attaattgcg
     4081 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4141 ggccaacgcg cggggagagg c
//
