LOCUS       sequence-328384-        6265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian cell expression vector for FLAG-tagged UPA-CARD of NLRP1. 
            The K1277E mutant abolishes interface III UPA-UPA contacts, DPP9 
            binding, and inflammasome activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6265)
  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 6265)
  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: pcDNA 3.1 LIC 6A NLRP1 UPA-CARD-FLAG K1277E
FEATURES             Location/Qualifiers
     source          1..6265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1722..1746)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1754..1777
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1818..1836)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1819..1836)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1856..1873)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1862..2086
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2132..2560
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2219..2238)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2429..2450
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2569..2589)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2574..2903
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2754..2889
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2816..2835
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2970..3764
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3024..3043)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3634..3653
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3938..4059
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3975..3994)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4029..4048
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4108..4124)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4108..4124)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4121..4143)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4132..4148
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4156..4162,4163..4180,4181..4186))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4201..4222
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4339..4356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4510..5098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4590..4609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5269..6060,6061..6129))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5892..5911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6130..6234)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccctacttcc aatccaatgc caccatgtcc cccttgggag tcctcctgaa
      961 aatgatccat aatgccctgc gcttcattcc cgtcacctct gtggtgttgc tttaccaccg
     1021 cgtccatcct gaggaagtca ccttccacct ctacctgatc ccaagtgact gctccattcg
     1081 gaaggccata gatgatctag aaatgaaatt ccagtttgtg cgaatccacg agccaccccc
     1141 gctgacccca ctttatatgg gctgtcgtta cactgtgtct gggtctggtt cagggatgct
     1201 ggaaatactc cccaaggaac tggagctctg ctatcgaagc cctggagaag accagctgtt
     1261 ctcggagttc tacgttggcc acttgggatc agggatcagg ctgcaagtga aagacaagaa
     1321 agatgagact ctggtgtggg aggccttggt gaaaccagga gatctcatgc ctgcaactac
     1381 tctgatccct ccagcccgca tagccgtacc ttcacctctg gatgccccgc agttgctgca
     1441 ctttgtggac cagtatcgag agcagctgat agcccgagtg acatcggtgg aggttgtctt
     1501 ggacaaactg catggacagg tgctgagcca ggagcagtac gagagggtgc tggctgagaa
     1561 cacgaggccc agccagatgc ggaagctgtt cagcttgagc cagtcctggg accggaagtg
     1621 caaagatgga ctctaccaag ccctgaagga gacccatcct cacctcatta tggaactctg
     1681 ggagaagggc agcaaaaagg gactcctgcc actcagcagc gacccagctt tcttgtacaa
     1741 agtggttgat atcgactaca aggacgatga cgacaagtag taataacatt ggaagtggat
     1801 aatctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg
     1861 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1921 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1981 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2041 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2101 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2161 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2221 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2281 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2341 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2401 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2461 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2521 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     2581 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2641 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2701 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     2761 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     2821 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     2881 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     2941 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3001 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3061 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3121 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3181 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3241 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3301 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3361 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3421 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3481 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3541 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3601 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3661 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3721 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3781 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     3841 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3901 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     3961 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4021 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     4081 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4141 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4201 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4261 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4321 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4381 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4441 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4501 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4561 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4621 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4681 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4741 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4801 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4861 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4921 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4981 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5041 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5101 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5161 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5221 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5281 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5341 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5401 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5461 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5521 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5581 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5641 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5701 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5761 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5821 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5881 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5941 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6001 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6061 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6121 aatactcata ctcttccttt ttcattatta ttgaagcatt tatcagggtt attgtctcat
     6181 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6241 tccccgaaaa gtgccacctg acgtc
//
