LOCUS       sequence-325111-        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 Y527A
FEATURES             Location/Qualifiers
     source          1..6867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..34
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             169..963
                     /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(223..242)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     833..852
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1137..1258
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1174..1193)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1228..1247
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1307..1323)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1307..1323)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1320..1342)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1331..1347
                     /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(1355..1361,1362..1379,1380..1385))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1400..1421
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1538..1555)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1709..2297)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1789..1808)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2468..3259,3260..3328))
                     /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     3091..3110
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3329..3433)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3508..3527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3699..4078
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4079..4282
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4233..4253
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4327..4346
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4327..4345
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5158..5865
                     /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(5300..5318)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5568..5587
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(5884..5902)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5885..5902)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5922..5939)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5928..6152
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6198..6626
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6285..6304)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6495..6516
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6635..6655)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6640..6867,1..102)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6820..6867,1..88)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
       61 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
      121 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
      181 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
      241 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
      301 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
      361 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
      421 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
      481 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
      541 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
      601 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
      661 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
      721 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
      781 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
      841 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
      901 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
      961 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     1021 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     1081 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     1141 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     1201 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     1261 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     1321 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     1381 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     1441 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1501 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1561 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1621 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1681 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1741 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1801 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1861 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1921 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1981 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2041 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2101 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2161 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2221 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2281 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2341 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2401 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2461 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2521 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2581 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2641 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2701 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2761 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     2821 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     2881 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     2941 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3001 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3061 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3121 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3181 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3241 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3301 gggcgacacg gaaatgttga atactcatac tcttcctttt tcattattat tgaagcattt
     3361 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3421 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     3481 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3541 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     3601 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     3661 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta
     3721 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     3781 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     3841 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     3901 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     3961 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     4021 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     4081 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     4141 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     4201 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     4261 ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt actggcttat
     4321 cgaaattaat acgactcact atagggagac ccaagcttgg taccgagctc ggatccctac
     4381 ttccaatcca atgccaccat gtctctgatg ggcatcctgc tgcggatcgc cagtgggact
     4441 cgcctctcca tccccatcac ttccaacaca ttgatctatt atcaccccca ccccgaagat
     4501 attaagttcc acttgtacct tgtccccagc gacgccttgc taacaaaggc gatagatgat
     4561 gaggaagatc gcttccatgg tgtgcgcctg cagacttcgc ccccaatgga acccctgaac
     4621 tttggttcca gttatattgt gtctaattct gctaacctga aagtaatgcc caaggagttg
     4681 aaattgtcct acaggagccc tggagaaatt cagcacttct caaaattcta tgctgggcag
     4741 atgaaggaac ccattcaact tgagattact gaaaaaagac atgggacttt ggtgtgggat
     4801 actgaggtga agccagtgga tctccagctt gtagctgcat cagcccctcc tcctttctca
     4861 ggtgcagcct ttgtgaagga gaaccaccgg caactccaag ccaggatggg ggacctgaaa
     4921 ggggtgctcg atgatctcca agacaatgag gttcttactg agaatgagaa ggagctggtg
     4981 gagcaggaaa agacacggca gagcaagaat gaggccttgc tgagcatggt ggagaagaaa
     5041 ggggacctgg ccctggacgt gctcttcaga agcattagtg aaagggaccc tgccctcgtg
     5101 tcctatctta gacagcagaa tggcattggt agtgggagca acggcagcag cggatccgtg
     5161 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     5221 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     5281 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     5341 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     5401 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     5461 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctcca ggacggcgag
     5521 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     5581 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     5641 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     5701 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     5761 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     5821 gagggccgcc actccaccgg cggcatggac gagctgtaca agtagtaatc tagagggccc
     5881 tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag
     5941 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     6001 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     6061 ttctattctg gggggtgggg tggggcagta cagcaagggg gaggattggg aagacaatag
     6121 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     6181 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     6241 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     6301 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     6361 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     6421 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6481 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     6541 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6601 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     6661 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     6721 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     6781 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     6841 agttccgccc attctccgcc ccatggc
//
