LOCUS       sequence-332710-        4172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR221 for Gateway shuttling. Contains CARD8 with a site III 
            mutation (L368G) with sgRNA binding site mutations .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4172)
  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 4172)
  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 L368G
FEATURES             Location/Qualifiers
     source          1..4172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     78..97
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     342..359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(518..545)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(637..723)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     772..794
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     786..803
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     787..803
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    819..918
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(2536..2635)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2652..2671)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2653..2671)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2676..2692)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2676..2692)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2794..2813)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2805..3614
                     /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(2872..2891)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(3761..4172,1..177)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
       61 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
      121 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
      181 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tggccttttg
      241 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      301 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      361 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
      421 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata
      481 cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc
      541 cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg
      601 ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt
      661 caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat
      721 ttgatgcctg gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac
      781 gacgttgtaa aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac
      841 tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt
      901 tgtacaaaaa agcaggcttc gccatggaaa aaaaggagtg tccagaaaag agtagcagca
      961 gtgaggaaga gctgccgaga cgggacagtg gatccagtag gaacatagat gcatccaaac
     1021 tcattagact acaaggatca cggaaactgt tggttgacaa tagcatacgg gaactgcaat
     1081 acacaaaaac tggaattttt tttcaggctg aggcctgtgt gacaaatgat acggtataca
     1141 gggagctacc ctgtgtttct gagacccttt gtgacatctc acattttttc caagaagatg
     1201 atgagacaga ggcagagcca ttattgttcc gtgctgttcc tgagtgtcaa ctatctgggg
     1261 gggacattcc cagtgtatca gaagagcagg aatcttcaga gggacaagat tcaggagaca
     1321 tttgctcaga agagaatcaa atagtttcct cttatgcttc taaagtctgt tttgagatcg
     1381 aagaagatta taaaaatcgt cagtttctgg ggcctgaagg aaatgtggat gttgagttga
     1441 ttgataagag cacaaacaga tacagcgttt ggttccccac tgctggctgg tatctgtggt
     1501 cagccacagg cctcggcttc ctggtaaggg atgaggtcac agttaccatc gccttcggca
     1561 gctggagtca gcacctggcc ctggacctgc agcaccatga acagtggctg gtgggcggcc
     1621 ccttgtttga tgtcactgca gagccagagg aggctgtcgc cgaaatccac ctcccccact
     1681 tcatctccct ccaagcaggt gaggtggacg tctcctggtt tctcgttgcc cattttaaga
     1741 atgaagggat ggtcctggag catccagccc gggtggagcc tttctatgct gtcctggaaa
     1801 gccccagctt ctctctgatg ggcatcctgc tgcggatcgc cagtgggact cgcctctcca
     1861 tccccatcac ttccaacaca ttgatctatt atcaccccca ccccgaagat attaagttcc
     1921 acttgtacct tgtccccagc gacgccttgc taacaaaggc gatagatgat gaggaagatc
     1981 gcttccatgg tgtgcgcctg cagacttcgc ccccaatgga acccgggaac tttggttcca
     2041 gttatattgt gtctaattct gctaacctga aagtaatgcc caaggagttg aaattgtcct
     2101 acaggagccc tggagaaatt cagcacttct caaaattcta tgctgggcag atgaaggaac
     2161 ccattcaact tgagattact gaaaaaagac atgggacttt ggtgtgggat actgaggtga
     2221 agccagtgga tctccagctt gtagctgcat cagcccctcc tcctttctca ggtgcagcct
     2281 ttgtgaagga gaaccaccgg caactccaag ccaggatggg ggacctgaaa ggggtgctcg
     2341 atgatctcca agacaatgag gttcttactg agaatgagaa ggagctggtg gagcaggaaa
     2401 agacacggca gagcaagaat gaggccttgc tgagcatggt ggagaagaaa ggggacctgg
     2461 ccctggacgt gctcttcaga agcattagtg aaagggaccc ttacctcgtg tcctatctta
     2521 gacagcagaa tttggaccca gctttcttgt acaaagttgg cattataaga aagcattgct
     2581 tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc
     2641 cagctgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct
     2701 ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa
     2761 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     2821 gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     2881 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg
     2941 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     3001 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     3061 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc
     3121 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     3181 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     3241 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     3301 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     3361 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     3421 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     3481 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     3541 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     3601 atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga
     3661 cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc
     3721 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3781 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3841 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3901 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3961 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4021 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4081 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4141 tacagcgtga gctatgagaa agcgccacgc tt
//
