LOCUS       sequence-332693-        4161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR221 for Gateway shuttling. Contains CARD8 with a site III 
            mutation (F405G) 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 F405G
FEATURES             Location/Qualifiers
     source          1..4161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(169..196)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(288..374)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     423..445
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     437..454
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     438..454
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    470..569
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(2187..2286)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2303..2322)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2304..2322)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2327..2343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2327..2343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2445..2464)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2456..3265
                     /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(2523..2542)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3412..4000
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3901..3920
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(4154..4161,1..10)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
       61 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      121 cgcaattaat acgcgtaccg ctagccagga agagtttgta gaaacgcaaa aaggccatcc
      181 gtcaggatgg ccttctgctt agtttgatgc ctggcagttt atggcgggcg tcctgcccgc
      241 caccctccgg gccgttgctt cacaacgttc aaatccgctc ccggcggatt tgtcctactc
      301 aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttc cgactgagcc
      361 tttcgtttta tttgatgcct ggcagttccc tactctcgcg ttaacgctag catggatgtt
      421 ttcccagtca cgacgttgta aaacgacggc cagtcttaag ctcgggcccc aaataatgat
      481 tttattttga ctgatagtga cctgttcgtt gcaacaaatt gatgagcaat gcttttttat
      541 aatgccaact ttgtacaaaa aagcaggctt cgccatggaa aaaaaggagt gtccagaaaa
      601 gagtagcagc agtgaggaag agctgccgag acgggacagt ggatccagta ggaacataga
      661 tgcatccaaa ctcattagac tacaaggatc acggaaactg ttggttgaca atagcatacg
      721 ggaactgcaa tacacaaaaa ctggaatttt ttttcaggct gaggcctgtg tgacaaatga
      781 tacggtatac agggagctac cctgtgtttc tgagaccctt tgtgacatct cacatttttt
      841 ccaagaagat gatgagacag aggcagagcc attattgttc cgtgctgttc ctgagtgtca
      901 actatctggg ggggacattc ccagtgtatc agaagagcag gaatcttcag agggacaaga
      961 ttcaggagac atttgctcag aagagaatca aatagtttcc tcttatgctt ctaaagtctg
     1021 ttttgagatc gaagaagatt ataaaaatcg tcagtttctg gggcctgaag gaaatgtgga
     1081 tgttgagttg attgataaga gcacaaacag atacagcgtt tggttcccca ctgctggctg
     1141 gtatctgtgg tcagccacag gcctcggctt cctggtaagg gatgaggtca cagttaccat
     1201 cgccttcggc agctggagtc agcacctggc cctggacctg cagcaccatg aacagtggct
     1261 ggtgggcggc cccttgtttg atgtcactgc agagccagag gaggctgtcg ccgaaatcca
     1321 cctcccccac ttcatctccc tccaagcagg tgaggtggac gtctcctggt ttctcgttgc
     1381 ccattttaag aatgaaggga tggtcctgga gcatccagcc cgggtggagc ctttctatgc
     1441 tgtcctggaa agccccagct tctctctgat gggcatcctg ctgcggatcg ccagtgggac
     1501 tcgcctctcc atccccatca cttccaacac attgatctat tatcaccccc accccgaaga
     1561 tattaagttc cacttgtacc ttgtccccag cgacgccttg ctaacaaagg cgatagatga
     1621 tgaggaagat cgcttccatg gtgtgcgcct gcagacttcg cccccaatgg aacccctgaa
     1681 ctttggttcc agttatattg tgtctaattc tgctaacctg aaagtaatgc ccaaggagtt
     1741 gaaattgtcc tacaggagcc ctggagaaat tcagcacttc tcaaaaggct atgctgggca
     1801 gatgaaggaa cccattcaac ttgagattac tgaaaaaaga catgggactt tggtgtggga
     1861 tactgaggtg aagccagtgg atctccagct tgtagctgca tcagcccctc ctcctttctc
     1921 aggtgcagcc tttgtgaagg agaaccaccg gcaactccaa gccaggatgg gggacctgaa
     1981 aggggtgctc gatgatctcc aagacaatga ggttcttact gagaatgaga aggagctggt
     2041 ggagcaggaa aagacacggc agagcaagaa tgaggccttg ctgagcatgg tggagaagaa
     2101 aggggacctg gccctggacg tgctcttcag aagcattagt gaaagggacc cttacctcgt
     2161 gtcctatctt agacagcaga atttggaccc agctttcttg tacaaagttg gcattataag
     2221 aaagcattgc ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat
     2281 tatttgccat ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc
     2341 ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     2401 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     2461 ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata
     2521 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta
     2581 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     2641 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     2701 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa
     2761 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     2821 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     2881 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     2941 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     3001 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     3061 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     3121 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     3181 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     3241 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag
     3301 cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagtta
     3361 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3421 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3481 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3541 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3601 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3661 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3721 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3781 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3841 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3901 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3961 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4021 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4081 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4141 aacgaccgag cgcagcgagt c
//
