LOCUS       sequence-325127-        6867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding CARD8 UPA-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 6867)
  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 6867)
  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 CARD8 UPA-CARD-mCherry R464E
FEATURES             Location/Qualifiers
     source          1..6867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1460..2167
                     /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(1602..1620)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1870..1889
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(2186..2204)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2187..2204)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2224..2241)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2230..2454
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2500..2928
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2587..2606)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2797..2818
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2937..2957)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2942..3271
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3122..3257
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3184..3203
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3338..4132
                     /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(3392..3411)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4002..4021
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4306..4427
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4343..4362)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4397..4416
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4476..4492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4476..4492)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4489..4511)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4500..4516
                     /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(4524..4530,4531..4548,4549..4554))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4569..4590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4707..4724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4878..5466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4958..4977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5637..6428,6429..6497))
                     /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     6260..6279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6498..6602)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6677..6696)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccct acttccaatc caatgccacc atgtctctga tgggcatcct
      721 gctgcggatc gccagtggga ctcgcctctc catccccatc acttccaaca cattgatcta
      781 ttatcacccc caccccgaag atattaagtt ccacttgtac cttgtcccca gcgacgcctt
      841 gctaacaaag gcgatagatg atgaggaaga tcgcttccat ggtgtgcgcc tgcagacttc
      901 gcccccaatg gaacccctga actttggttc cagttatatt gtgtctaatt ctgctaacct
      961 gaaagtaatg cccaaggagt tgaaattgtc ctacaggagc cctggagaaa ttcagcactt
     1021 ctcaaaattc tatgctgggc agatgaagga acccattcaa cttgagatta ctgaaaaaag
     1081 acatgggact ttggtgtggg atactgaggt gaagccagtg gatctccagc ttgtagctgc
     1141 atcagcccct cctcctttct caggtgcagc ctttgtgaag gagaaccacc ggcaactcca
     1201 agccgagatg ggggacctga aaggggtgct cgatgatctc caagacaatg aggttcttac
     1261 tgagaatgag aaggagctgg tggagcagga aaagacacgg cagagcaaga atgaggcctt
     1321 gctgagcatg gtggagaaga aaggggacct ggccctggac gtgctcttca gaagcattag
     1381 tgaaagggac ccttacctcg tgtcctatct tagacagcag aatggcattg gtagtgggag
     1441 caacggcagc agcggatccg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
     1501 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     1561 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     1621 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     1681 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     1741 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     1801 ctcctccctc caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     1861 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     1921 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     1981 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     2041 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     2101 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     2161 caagtagtaa tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat
     2221 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2281 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2341 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2401 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     2461 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     2521 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2581 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2641 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2701 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2761 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2821 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2881 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     2941 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3001 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3061 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3121 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3181 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     3241 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     3301 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     3361 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3421 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3481 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     3541 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3601 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3661 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3721 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3781 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3841 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     3901 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3961 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4021 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4081 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4141 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     4201 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4261 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4321 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4381 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4441 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4501 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4561 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4621 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4681 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4741 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4801 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4861 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4921 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4981 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5041 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5101 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5161 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5221 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5281 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5341 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5401 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5461 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5521 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5581 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5641 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5701 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5761 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5821 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5881 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5941 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6001 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6061 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6121 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6181 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6241 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6301 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6361 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6421 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6481 aaatgttgaa tactcatact cttccttttt cattattatt gaagcattta tcagggttat
     6541 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6601 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     6661 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     6721 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     6781 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     6841 gcgatgtacg ggccagatat acgcgtt
//
