LOCUS       sequence-324622-        6447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding codon-optimized human NLRP1 
            CARD (dimer interface 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     62..83
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(202..222)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        207..536
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      387..522
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     449..468
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             603..1397
                     /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(657..676)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1267..1286
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1571..1692
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1608..1627)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1662..1681
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1741..1757)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1741..1757)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1754..1776)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1765..1781
                     /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(1789..1795,1796..1813,1814..1819))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1834..1855
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1972..1989)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2143..2731)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2223..2242)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2902..3693,3694..3762))
                     /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     3525..3544
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3763..3867)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3942..3961)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4133..4512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4513..4716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4667..4687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4761..4780
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4761..4779
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4827..4836
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5172..5879
                     /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(5314..5332)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5582..5601
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(5898..5916)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5899..5916)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5936..5953)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5942..6166
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6212..6447,1..193)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6299..6318)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
       61 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
      121 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
      181 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
      241 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
      301 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
      361 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
      421 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
      481 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
      541 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
      601 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
      661 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
      721 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
      781 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
      841 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
      901 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
      961 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     1021 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     1081 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     1141 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     1201 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     1261 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     1321 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     1381 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     1441 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     1501 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     1561 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1621 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     1681 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     1741 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     1801 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     1861 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1921 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1981 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2041 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2101 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2161 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2221 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2281 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2341 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2401 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2461 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2521 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2581 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2641 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2701 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2761 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2821 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2881 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2941 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3001 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3061 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3121 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3181 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3241 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3301 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     3361 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3421 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3481 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3541 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3601 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3661 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3721 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcatta
     3781 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3841 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     3901 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     3961 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     4021 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     4081 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
     4141 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     4201 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     4261 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     4321 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     4381 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     4441 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     4501 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     4561 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     4621 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     4681 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
     4741 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc ttggtaccga
     4801 gctcggatcc ctacttccaa tccaatgcca ccatggacgc tcctcagctg ctgcatttcg
     4861 tggaccagta ccgggagcag ctgatcgcta gagtgacctc cgtggaagtg gtgctggaca
     4921 agctgcacgg acaggtgctg agtcaggagc agtacgagag ggtgctggca gagaacacca
     4981 ggcctagcca gatgcggaag ctgttcagcc tgagccagtc ttgggacagg aagtgcaagg
     5041 acggcctgta ccaggctctg aaggagacac acccccacct gatcgcggag ctctgggaga
     5101 agggaagcaa gaagggcctg ctgcctctgt ctagcggcat tggtagtggg agcaacggca
     5161 gcagcggatc cgtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     5221 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     5281 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     5341 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     5401 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     5461 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     5521 tccaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     5581 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     5641 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     5701 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     5761 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     5821 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtagt
     5881 aatctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg
     5941 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     6001 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     6061 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     6121 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     6181 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     6241 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     6301 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     6361 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     6421 cttgattagg gtgatggttc acgtagt
//
