LOCUS       sequence-324618-        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"
     primer_bind     complement(21..40)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     75..94
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(154..170)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(154..170)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(167..189)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    178..194
                     /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(202..208,209..226,227..232))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    247..268
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(385..402)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(556..1144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(636..655)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1315..2106,2107..2175))
                     /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     1938..1957
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2176..2280)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2355..2374)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2546..2925
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2926..3129
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3080..3100
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3174..3193
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3174..3192
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3240..3249
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3585..4292
                     /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(3727..3745)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3995..4014
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(4311..4329)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4312..4329)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4349..4366)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4355..4579
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4625..5053
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4712..4731)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4922..4943
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5062..5082)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5067..5396
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5247..5382
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5309..5328
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5463..6257
                     /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(5517..5536)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6127..6146
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    join(6431..6447,1..105)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
       61 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
      121 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
      181 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
      241 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
      301 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
      361 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
      421 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      481 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      541 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      601 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      661 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      721 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      781 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      841 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      901 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      961 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     1021 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1081 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1141 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1201 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     1261 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     1321 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1381 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1441 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1501 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1561 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1621 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1681 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1741 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1801 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1861 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1921 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1981 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     2041 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     2101 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     2161 atgttgaata ctcatactct tcctttttca ttattattga agcatttatc agggttattg
     2221 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     2281 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     2341 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     2401 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     2461 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     2521 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     2581 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     2641 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     2701 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     2761 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     2821 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     2881 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     2941 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     3001 attgacgtca ataggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     3061 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     3121 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
     3181 actcactata gggagaccca agcttggtac cgagctcgga tccctacttc caatccaatg
     3241 ccaccatgga cgctcctcag ctgctgcatt tcgtggacca gtaccgggag cagctgatcg
     3301 ctagagtgac ctccgtggaa gtggtgctgg acaagctgca cggacaggtg ctgagtcagg
     3361 agcagtaccg gagggtgctg gcagagaaca ccaggcctag ccagatgcgg aagctgttca
     3421 gcctgagcca gtcttgggac aggaagtgca aggacggcct gtaccaggct ctgaaggaga
     3481 cacaccccca cctgatcatg gagctctggg agaagggaag caagaagggc ctgctgcctc
     3541 tgtctagcgg cattggtagt gggagcaacg gcagcagcgg atccgtgagc aagggcgagg
     3601 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     3661 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     3721 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     3781 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     3841 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     3901 gcggcgtggt gaccgtgacc caggactcct ccctccagga cggcgagttc atctacaagg
     3961 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     4021 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     4081 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     4141 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     4201 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     4261 ccaccggcgg catggacgag ctgtacaagt agtaatctag agggccctat tctatagtgt
     4321 cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     4381 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4441 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4501 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     4561 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     4621 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4681 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4741 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4801 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4861 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4921 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     4981 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5041 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     5101 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     5161 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     5221 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     5281 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     5341 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     5401 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     5461 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     5521 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     5581 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     5641 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     5701 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     5761 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     5821 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     5881 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     5941 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     6001 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     6061 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     6121 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     6181 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     6241 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     6301 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     6361 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     6421 cgcccacccc aacttgttta ttgcagc
//
