LOCUS       sequence-328304-        3564 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR221 encoding ubiquitin-tagged NLRP1-CT to generate the native 
            S1213 N-terminus, with the P1214R patient mutation (Grandemange et 
            al., 2017) (no stop).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3564)
  AUTHORS   Hollingsworth LR, Sharif H, Griswold AR, Fontana P, Mintseris J, 
            Dagbay KB, Paulo JA, Gygi SP, Bachovchin DA, Wu H
  TITLE     DPP9 sequesters the C terminus of NLRP1 to repress inflammasome 
            activation.
  JOURNAL   Nature. 2021 Mar 17. pii: 10.1038/s41586-021-03350-4. doi: 
            10.1038/s41586-021-03350-4.
  PUBMED    33731932
REFERENCE   2  (bases 1 to 3564)
  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"; journalName: "Nature. 
            2021 Mar 17. pii: 10.1038/s41586-021-03350-4. doi: 
            10.1038/s41586-021-03350-4."
COMMENT     Sequence Label: pDONR221 Ub-NLRP1 S1213 P1214R
FEATURES             Location/Qualifiers
     source          1..3564
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             677..904
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     protein_bind    complement(1689..1788)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1805..1824)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1806..1824)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1829..1845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1829..1845)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1947..1966)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1958..2767
                     /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(2025..2044)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2914..3502
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3403..3422
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggcttc gccaccatgc agatcttcgt caagacgtta accggtaaaa ccataactct
      721 ggaagttgaa ccatccgata ccatcgaaaa cgttaaggct aaaattcaag acaaggaagg
      781 aattccacct gatcaacaaa gattgatctt tgccggtaag cagctcgagg acggtagaac
      841 gctgtctgat tacaacattc agaaggagtc gaccttacat cttgtcttaa gactaagagg
      901 tggttcccgc ttgggagtcc tcctgaaaat gatccataat gccctgcgct tcattcccgt
      961 cacctctgtg gtgttgcttt accaccgcgt ccatcctgag gaagtcacct tccacctcta
     1021 cctgatccca agtgactgct ccattcggaa ggccatagat gatctagaaa tgaaattcca
     1081 gtttgtgcga atccacaagc cacccccgct gaccccactt tatatgggct gtcgttacac
     1141 tgtgtctggg tctggttcag ggatgctgga aatactcccc aaggaactgg agctctgcta
     1201 tcgaagccct ggagaagacc agctgttctc ggagttctac gttggccact tgggatcagg
     1261 gatcaggctg caagtgaaag acaagaaaga tgagactctg gtgtgggagg ccttggtgaa
     1321 accaggagat ctcatgcctg caactactct gatccctcca gcccgcatag ccgtaccttc
     1381 acctctggat gccccgcagt tgctgcactt tgtggaccag tatcgagagc agctgatagc
     1441 ccgagtgaca tcggtggagg ttgtcttgga caaactgcat ggacaggtgc tgagccagga
     1501 gcagtacgag agggtgctgg ctgagaacac gaggcccagc cagatgcgga agctgttcag
     1561 cttgagccag tcctgggacc ggaagtgcaa agatggactc taccaagccc tgaaggagac
     1621 ccatcctcac ctcattatgg aactctggga gaagggcagc aaaaagggac tcctgccact
     1681 cagcagcgac ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat
     1741 ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctga
     1801 tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt
     1861 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa
     1921 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     1981 gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     2041 cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc
     2101 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     2161 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     2221 tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt
     2281 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     2341 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     2401 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     2461 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     2521 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     2581 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     2641 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     2701 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     2761 tttctaatca gaattggtta attggttgta acactggcag agcattacgc tgacttgacg
     2821 ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc
     2881 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2941 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3001 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3061 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3121 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3181 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3241 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3301 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3361 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3421 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3481 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3541 ttgctggcct tttgctcaca tgtt
//
