LOCUS       sequence-322631-        6265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding human NLRP1 UPA-CARD 
            (filament-deficient mutant) with a C-terminal FLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6265)
  AUTHORS   Robert Hollingsworth L, David L, Li Y, Griswold AR, Ruan J, Sharif 
            H, Fontana P, Orth-He EL, Fu TM, Bachovchin DA, Wu H
  TITLE     Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 
            inflammasomes.
  JOURNAL   Nat Commun. 2021 Jan 8;12(1):189. doi: 10.1038/s41467-020-20320-y.
  PUBMED    33420033
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: "Nat 
            Commun."; date: "2021-01-8"; volume: "12(1):189. doi"; pages: " 
            10.1038/s41467-020-20320-y"
COMMENT     Sequence Label: pcDNA3.1 6A NLRP1 UPA-CARD-FLAG R1392E
FEATURES             Location/Qualifiers
     source          1..6265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             87..881
                     /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(141..160)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     751..770
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1055..1176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1092..1111)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1146..1165
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1225..1241)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1225..1241)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1238..1260)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1249..1265
                     /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(1273..1279,1280..1297,1298..1303))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1318..1339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1456..1473)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1627..2215)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1707..1726)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2386..3177,3178..3246))
                     /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     3009..3028
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3247..3351)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3426..3445)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3617..3996
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3997..4200
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4151..4171
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4245..4264
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4245..4263
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(5104..5128)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5136..5159
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(5200..5218)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5201..5218)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5238..5255)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5244..5468
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5514..5942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5601..5620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5811..5832
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5951..5971)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(5956..6265,1..20)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6136..6265,1..6)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6198..6217
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
       61 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
      121 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
      181 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
      241 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
      301 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
      361 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
      421 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
      481 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
      541 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
      601 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
      661 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
      721 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
      781 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
      841 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
      901 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
      961 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     1021 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     1081 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     1141 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     1201 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1261 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1321 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1381 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1441 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     1501 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     1561 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     1621 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     1681 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1741 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     1801 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     1861 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     1921 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     1981 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2041 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2101 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2161 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2221 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2281 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     2341 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     2401 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     2461 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     2521 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     2581 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     2641 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     2701 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     2761 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     2821 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     2881 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     2941 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3001 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3061 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3121 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     3181 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     3241 actcatactc ttcctttttc attattattg aagcatttat cagggttatt gtctcatgag
     3301 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     3361 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     3421 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     3481 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     3541 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     3601 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     3661 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     3721 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     3781 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     3841 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     3901 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     3961 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     4021 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     4081 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     4141 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     4201 ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat
     4261 agggagaccc aagcttggta ccgagctcgg atccctactt ccaatccaat gccaccatgt
     4321 cccccttggg agtcctcctg aaaatgatcc ataatgccct gcgcttcatt cccgtcacct
     4381 ctgtggtgtt gctttaccac cgcgtccatc ctgaggaagt caccttccac ctctacctga
     4441 tcccaagtga ctgctccatt cggaaggcca tagatgatct agaaatgaaa ttccagtttg
     4501 tgcgaatcca caagccaccc ccgctgaccc cactttatat gggctgtcgt tacactgtgt
     4561 ctgggtctgg ttcagggatg ctggaaatac tccccaagga actggagctc tgctatcgaa
     4621 gccctggaga agaccagctg ttctcggagt tctacgttgg ccacttggga tcagggatca
     4681 ggctgcaagt gaaagacaag aaagatgaga ctctggtgtg ggaggccttg gtgaaaccag
     4741 gagatctcat gcctgcaact actctgatcc ctccagcccg catagccgta ccttcacctc
     4801 tggatgcccc gcagttgctg cactttgtgg accagtatcg agagcagctg atagccgaag
     4861 tgacatcggt ggaggttgtc ttggacaaac tgcatggaca ggtgctgagc caggagcagt
     4921 acgagagggt gctggctgag aacacgaggc ccagccagat gcggaagctg ttcagcttga
     4981 gccagtcctg ggaccggaag tgcaaagatg gactctacca agccctgaag gagacccatc
     5041 ctcacctcat tatggaactc tgggagaagg gcagcaaaaa gggactcctg ccactcagca
     5101 gcgacccagc tttcttgtac aaagtggttg atatcgacta caaggacgat gacgacaagt
     5161 agtaataaca ttggaagtgg ataatctaga gggccctatt ctatagtgtc acctaaatgc
     5221 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     5281 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     5341 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     5401 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     5461 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     5521 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     5581 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     5641 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     5701 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     5761 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5821 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     5881 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5941 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     6001 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     6061 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     6121 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     6181 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     6241 cagaagtagt gaggaggctt ttttg
//
