LOCUS       sequence-324619-        6447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding codon-optimized human NLRP1 
            CARD (filament-deficient mutation) with a C-terminal mCherry tag for
            imaging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6447)
  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 6447)
  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 6B codon-optimized NLRP1 CARD-mCherry...
FEATURES             Location/Qualifiers
     source          1..6447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             53..847
                     /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(107..126)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     717..736
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1021..1142
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1058..1077)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1112..1131
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1191..1207)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1191..1207)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1204..1226)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1215..1231
                     /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(1239..1245,1246..1263,1264..1269))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1284..1305
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1422..1439)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1593..2181)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1673..1692)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2352..3143,3144..3212))
                     /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     2975..2994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3213..3317)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3392..3411)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3583..3962
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3963..4166
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4117..4137
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4211..4230
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4211..4229
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4277..4286
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4622..5329
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4764..4782)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5032..5051
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(5348..5366)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5349..5366)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5386..5403)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5392..5616
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5662..6090
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5749..5768)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5959..5980
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6099..6119)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6104..6433
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6284..6419
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6346..6365
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
       61 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
      121 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
      181 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
      241 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
      301 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
      361 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
      421 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
      481 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
      541 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
      601 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
      661 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
      721 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
      781 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
      841 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
      901 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
      961 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     1021 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1081 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1141 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     1201 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     1261 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     1321 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1381 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     1441 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1501 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     1561 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     1621 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     1681 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     1741 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     1801 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     1861 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     1921 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     1981 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     2041 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2101 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     2161 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     2221 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     2281 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2341 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2401 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2461 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     2521 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     2581 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     2641 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     2701 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     2761 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     2821 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     2881 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     2941 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     3001 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3061 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3121 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     3181 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcatta ttattgaagc
     3241 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     3301 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     3361 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3421 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     3481 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     3541 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     3601 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     3661 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     3721 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     3781 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     3841 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     3901 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     3961 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     4021 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     4081 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     4141 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
     4201 ttatcgaaat taatacgact cactataggg agacccaagc ttggtaccga gctcggatcc
     4261 ctacttccaa tccaatgcca ccatggacgc tcctcagctg ctgcatttcg tggaccagta
     4321 ccgggagcag ctgatcgcta gagtgacctc cgtggaagtg gtgctggaca agctgcacgg
     4381 acaggtgctg agtcaggagc agtacgagag ggtgctggca gagaacacca ggcctagcca
     4441 gatggagaag ctgttcagcc tgagccagtc ttgggacagg aagtgcaagg acggcctgta
     4501 ccaggctctg aaggagacac acccccacct gatcatggag ctctgggaga agggaagcaa
     4561 gaagggcctg ctgcctctgt ctagcggcat tggtagtggg agcaacggca gcagcggatc
     4621 cgtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     4681 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     4741 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     4801 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     4861 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     4921 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tccaggacgg
     4981 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     5041 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     5101 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     5161 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     5221 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     5281 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtagt aatctagagg
     5341 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     5401 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     5461 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     5521 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     5581 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     5641 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     5701 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     5761 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5821 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     5881 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5941 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     6001 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     6061 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     6121 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     6181 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     6241 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     6301 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     6361 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     6421 aggcttttgc aaaaagctcc cgggagc
//
