LOCUS       sequence-324608-        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"
     promoter        complement(128..146)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(129..146)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(166..183)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    172..396
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      442..870
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(529..548)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     739..760
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(879..899)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        884..1213
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1064..1199
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1126..1145
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1280..2074
                     /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(1334..1353)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1944..1963
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2248..2369
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2285..2304)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2339..2358
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2418..2434)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2418..2434)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2431..2453)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2442..2458
                     /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(2466..2472,2473..2490,2491..2496))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2511..2532
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2649..2666)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2820..3408)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2900..2919)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3579..4370,4371..4439))
                     /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     4202..4221
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4440..4544)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4619..4638)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4810..5189
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5190..5393
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5344..5364
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5438..5457
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5438..5456
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5504..5513
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5849..6447,1..109)
                     /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(5991..6009)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6259..6278
                     /label=mCherry-F
                     /note="mCherry, forward primer"
ORIGIN
        1 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
       61 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtagt aatctagagg
      121 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
      181 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
      241 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
      301 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
      361 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
      421 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
      481 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
      541 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
      601 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
      661 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
      721 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
      781 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
      841 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
      901 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      961 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     1021 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     1081 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     1141 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     1201 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     1261 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     1321 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     1381 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     1441 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     1501 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     1561 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     1621 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     1681 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     1741 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     1801 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     1861 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     1921 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     1981 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     2041 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     2101 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     2161 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     2221 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     2281 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2341 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     2401 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2461 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2521 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2581 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2641 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2701 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2761 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2821 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2881 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2941 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3001 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3061 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3121 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3181 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3241 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3301 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3361 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3421 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3481 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3541 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3601 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3661 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3721 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3781 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3841 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3901 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3961 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4021 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4081 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4141 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4201 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4261 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4321 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4381 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4441 ctcttccttt ttcattatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4501 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4561 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     4621 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     4681 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     4741 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     4801 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     4861 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     4921 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     4981 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     5041 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     5101 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     5161 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     5221 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     5281 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     5341 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
     5401 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
     5461 cccaagcttg gtaccgagct cggatcccta cttccaatcc aatgccacca tggacgctcc
     5521 tcagctgctg catttcgtgg accagtaccg ggagcagctg atcgctgaag tgacctccgt
     5581 ggaagtggtg ctggacaagc tgcacggaca ggtgctgagt caggagcagt acgagagggt
     5641 gctggcagag aacaccaggc ctagccagat gcggaagctg ttcagcctga gccagtcttg
     5701 ggacaggaag tgcaaggacg gcctgtacca ggctctgaag gagacacacc cccacctgat
     5761 catggagctc tgggagaagg gaagcaagaa gggcctgctg cctctgtcta gcggcattgg
     5821 tagtgggagc aacggcagca gcggatccgt gagcaagggc gaggaggata acatggccat
     5881 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     5941 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     6001 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     6061 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     6121 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     6181 gacccaggac tcctccctcc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     6241 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc
     6301 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     6361 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     6421 gcagctgccc ggcgcctaca acgtcaa
//
