LOCUS       sequence-324611-        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(169..187)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(170..187)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(207..224)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    213..437
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      483..911
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(570..589)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     780..801
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(920..940)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        925..1254
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1105..1240
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1167..1186
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1321..2115
                     /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(1375..1394)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1985..2004
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2289..2410
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2326..2345)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2380..2399
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2459..2475)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2459..2475)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2472..2494)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2483..2499
                     /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(2507..2513,2514..2531,2532..2537))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2552..2573
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2690..2707)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2861..3449)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2941..2960)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3620..4411,4412..4480))
                     /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     4243..4262
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4481..4585)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4660..4679)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4851..5230
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5231..5434
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5385..5405
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5479..5498
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5479..5497
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5545..5554
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5890..6447,1..150)
                     /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(6032..6050)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6300..6319
                     /label=mCherry-F
                     /note="mCherry, forward primer"
ORIGIN
        1 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
       61 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
      121 accggcggca tggacgagct gtacaagtag taatctagag ggccctattc tatagtgtca
      181 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
      241 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
      301 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
      361 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
      421 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
      481 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
      541 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
      601 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
      661 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
      721 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
      781 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
      841 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
      901 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
      961 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     1021 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     1081 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     1141 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     1201 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     1261 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     1321 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     1381 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     1441 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     1501 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     1561 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     1621 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     1681 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     1741 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     1801 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     1861 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     1921 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     1981 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     2041 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     2101 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     2161 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     2221 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     2281 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     2341 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     2401 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     2461 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2521 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2581 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2641 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2701 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2761 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2821 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2881 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2941 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3001 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3061 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3121 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3181 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3241 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3301 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3361 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3421 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3481 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3541 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3601 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3661 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3721 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3781 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3841 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3901 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3961 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4021 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4081 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4141 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4201 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4261 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4321 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4381 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4441 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcattatt
     4501 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4561 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     4621 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     4681 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     4741 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     4801 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     4861 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     4921 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     4981 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     5041 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     5101 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     5161 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     5221 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     5281 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     5341 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     5401 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
     5461 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt ggtaccgagc
     5521 tcggatccct acttccaatc caatgccacc atggacgctc ctcagctgct gcatttcgtg
     5581 gaccagtacc gggagcagct gatcgctaga gtgacctccg tgcgagtggt gctggacaag
     5641 ctgcacggac aggtgctgag tcaggagcag tacgagaggg tgctggcaga gaacaccagg
     5701 cctagccaga tgcggaagct gttcagcctg agccagtctt gggacaggaa gtgcaaggac
     5761 ggcctgtacc aggctctgaa ggagacacac ccccacctga tcatggagct ctgggagaag
     5821 ggaagcaaga agggcctgct gcctctgtct agcggcattg gtagtgggag caacggcagc
     5881 agcggatccg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     5941 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     6001 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     6061 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     6121 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     6181 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctc
     6241 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     6301 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     6361 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     6421 gacgctgagg tcaagaccac ctacaag
//
