LOCUS       sequence-332714-        4161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR221 for Gateway shuttling. Contains CARD8 (no stop codon) 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
FEATURES             Location/Qualifiers
     source          1..4161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..46)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(200..788)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(280..299)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(935..1744)
                     /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     1658..1677
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1736..1755
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1857..1873
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1857..1873
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1878..1897
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1878..1896
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1914..2013
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(3631..3730)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3746..3763)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3746..3762)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3755..3777)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      3826..3912
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4004..4031
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
       61 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
      121 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
      181 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      241 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      301 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      361 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      421 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      481 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      541 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      601 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
      661 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
      721 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      781 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgacgcgt
      841 aactcacgtt aagggatttt ggtcatgagc ttgcgccgtc ccgtcaagtc agcgtaatgc
      901 tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat
      961 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     1021 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     1081 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     1141 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     1201 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     1261 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     1321 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     1381 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     1441 ttccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     1501 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     1561 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     1621 acaagcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     1681 ataaatcagc atccatgttg gaatttaatc gcggcctcga cgtttcccgt tgaatatggc
     1741 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata
     1801 tatttttatc ttgtgcaatg taacatcaga gattttgaga cacgggccag agctgccagg
     1861 aaacagctat gaccatgtaa tacgactcac tataggggat atcagctgga tggcaaataa
     1921 tgattttatt ttgactgata gtgacctgtt cgttgcaaca aattgataag caatgctttc
     1981 ttataatgcc aactttgtac aagaaagctg ggtccaaatt ctgctgtcta agataggaca
     2041 cgaggtaagg gtccctttca ctaatgcttc tgaagagcac gtccagggcc aggtcccctt
     2101 tcttctccac catgctcagc aaggcctcat tcttgctctg ccgtgtcttt tcctgctcca
     2161 ccagctcctt ctcattctca gtaagaacct cattgtcttg gagatcatcg agcacccctt
     2221 tcaggtcccc catcctggct tggagttgcc ggtggttctc cttcacaaag gctgcacctg
     2281 agaaaggagg aggggctgat gcagctacaa gctggagatc cactggcttc acctcagtat
     2341 cccacaccaa agtcccatgt cttttttcag taatctcaag ttgaatgggt tccttcatct
     2401 gcccagcata gaattttgag aagtgctgaa tttctccagg gctcctgtag gacaatttca
     2461 actccttggg cattactttc aggttagcag aattagacac aatataactg gaaccaaagt
     2521 tcaggggttc cattgggggc gaagtctgca ggcgcacacc atggaagcga tcttcctcat
     2581 catctatcgc ctttgttagc aaggcgtcgc tggggacaag gtacaagtgg aacttaatat
     2641 cttcggggtg ggggtgataa tagatcaatg tgttggaagt gatggggatg gagaggcgag
     2701 tcccactggc gatccgcagc aggatgccca tcagagagaa gctggggctt tccaggacag
     2761 catagaaagg ctccacccgg gctggatgct ccaggaccat cccttcattc ttaaaatggg
     2821 caacgagaaa ccaggagacg tccacctcac ctgcttggag ggagatgaag tgggggaggt
     2881 ggatttcggc gacagcctcc tctggctctg cagtgacatc aaacaagggg ccgcccacca
     2941 gccactgttc atggtgctgc aggtccaggg ccaggtgctg actccagctg ccgaaggcga
     3001 tggtaactgt gacctcatcc cttaccagga agccgaggcc tgtggctgac cacagatacc
     3061 agccagcagt ggggaaccaa acgctgtatc tgtttgtgct cttatcaatc aactcaacat
     3121 ccacatttcc ttcaggcccc agaaactgac gatttttata atcttcttcg atctcaaaac
     3181 agactttaga agcataagag gaaactattt gattctcttc tgagcaaatg tctcctgaat
     3241 cttgtccctc tgaagattcc tgctcttctg atacactggg aatgtccccc ccagatagtt
     3301 gacactcagg aacagcacgg aacaataatg gctctgcctc tgtctcatca tcttcttgga
     3361 aaaaatgtga gatgtcacaa agggtctcag aaacacaggg tagctccctg tataccgtat
     3421 catttgtcac acaggcctca gcctgaaaaa aaattccagt ttttgtgtat tgcagttccc
     3481 gtatgctatt gtcaaccaac agtttccgtg atccttgtag tctaatgagt ttggatgcat
     3541 ctatgttcct actggatcca ctgtcccgtc tcggcagctc ttcctcactg ctgctactct
     3601 tttctggaca ctcctttttt tccatggcga agcctgcttt tttgtacaaa gttggcatta
     3661 taaaaaagca ttgctcatca atttgttgca acgaacaggt cactatcagt caaaataaaa
     3721 tcattatttg gggcccgagc ttaagactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3781 acatccatgc tagcgttaac gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     3841 gctcagtcgg aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     3901 agtaggacaa atccgccggg agcggatttg aacgttgtga agcaacggcc cggagggtgg
     3961 cgggcaggac gcccgccata aactgccagg catcaaacta agcagaaggc catcctgacg
     4021 gatggccttt ttgcgtttct acaaactctt cctggctagc ggtacgcgta ttaattgcgt
     4081 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4141 gccaacgcgc ggggagaggc g
//
