LOCUS       sequence-332704-        4161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR221 for Gateway shuttling. Contains CARD8 that is 
            autoprocessing-deficient (S297A) 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 S297A
FEATURES             Location/Qualifiers
     source          1..4161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(547..574)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(666..752)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     801..823
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     815..832
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     816..832
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    848..947
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(2565..2664)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2681..2700)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2682..2700)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2705..2721)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2705..2721)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2823..2842)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2834..3643
                     /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(2901..2920)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(3790..4161,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatac gcgtaccgct agccaggaag agtttgtaga
      541 aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat
      601 ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc
      661 ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc
      721 cagtcttccg actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcgtt
      781 aacgctagca tggatgtttt cccagtcacg acgttgtaaa acgacggcca gtcttaagct
      841 cgggccccaa ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga
      901 tgagcaatgc ttttttataa tgccaacttt gtacaaaaaa gcaggcttcg ccatggaaaa
      961 aaaggagtgt ccagaaaaga gtagcagcag tgaggaagag ctgccgagac gggacagtgg
     1021 atccagtagg aacatagatg catccaaact cattagacta caaggatcac ggaaactgtt
     1081 ggttgacaat agcatacggg aactgcaata cacaaaaact ggaatttttt ttcaggctga
     1141 ggcctgtgtg acaaatgata cggtatacag ggagctaccc tgtgtttctg agaccctttg
     1201 tgacatctca cattttttcc aagaagatga tgagacagag gcagagccat tattgttccg
     1261 tgctgttcct gagtgtcaac tatctggggg ggacattccc agtgtatcag aagagcagga
     1321 atcttcagag ggacaagatt caggagacat ttgctcagaa gagaatcaaa tagtttcctc
     1381 ttatgcttct aaagtctgtt ttgagatcga agaagattat aaaaatcgtc agtttctggg
     1441 gcctgaagga aatgtggatg ttgagttgat tgataagagc acaaacagat acagcgtttg
     1501 gttccccact gctggctggt atctgtggtc agccacaggc ctcggcttcc tggtaaggga
     1561 tgaggtcaca gttaccatcg ccttcggcag ctggagtcag cacctggccc tggacctgca
     1621 gcaccatgaa cagtggctgg tgggcggccc cttgtttgat gtcactgcag agccagagga
     1681 ggctgtcgcc gaaatccacc tcccccactt catctccctc caagcaggtg aggtggacgt
     1741 ctcctggttt ctcgttgccc attttaagaa tgaagggatg gtcctggagc atccagcccg
     1801 ggtggagcct ttctatgctg tcctggaaag ccccagcttc gccctgatgg gcatcctgct
     1861 gcggatcgcc agtgggactc gcctctccat ccccatcact tccaacacat tgatctatta
     1921 tcacccccac cccgaagata ttaagttcca cttgtacctt gtccccagcg acgccttgct
     1981 aacaaaggcg atagatgatg aggaagatcg cttccatggt gtgcgcctgc agacttcgcc
     2041 cccaatggaa cccctgaact ttggttccag ttatattgtg tctaattctg ctaacctgaa
     2101 agtaatgccc aaggagttga aattgtccta caggagccct ggagaaattc agcacttctc
     2161 aaaattctat gctgggcaga tgaaggaacc cattcaactt gagattactg aaaaaagaca
     2221 tgggactttg gtgtgggata ctgaggtgaa gccagtggat ctccagcttg tagctgcatc
     2281 agcccctcct cctttctcag gtgcagcctt tgtgaaggag aaccaccggc aactccaagc
     2341 caggatgggg gacctgaaag gggtgctcga tgatctccaa gacaatgagg ttcttactga
     2401 gaatgagaag gagctggtgg agcaggaaaa gacacggcag agcaagaatg aggccttgct
     2461 gagcatggtg gagaagaaag gggacctggc cctggacgtg ctcttcagaa gcattagtga
     2521 aagggaccct tacctcgtgt cctatcttag acagcagaat ttggacccag ctttcttgta
     2581 caaagttggc attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat
     2641 cagtcaaaat aaaatcatta tttgccatcc agctgatatc ccctatagtg agtcgtatta
     2701 catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct gatgttacat
     2761 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     2821 tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa
     2881 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
     2941 gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
     3001 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
     3061 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac
     3121 cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
     3181 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     3241 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa
     3301 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     3361 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     3421 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     3481 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     3541 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
     3601 gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
     3661 gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa
     3721 atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3781 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3841 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3901 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     3961 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4021 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4081 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4141 gcgaacgacc tacaccgaac t
//
