LOCUS       sequence-322630-        6265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding human NLRP1 UPA-CARD 
            (filament-deficient mutant) with a C-terminal FLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6265)
  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 6265)
  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 6A NLRP1 UPA-CARD-FLAG R1386E
FEATURES             Location/Qualifiers
     source          1..6265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(113..133)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        118..447
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      298..433
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     360..379
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             514..1308
                     /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(568..587)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1178..1197
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1482..1603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1519..1538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1573..1592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1652..1668)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1652..1668)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1665..1687)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1676..1692
                     /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(1700..1706,1707..1724,1725..1730))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1745..1766
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1883..1900)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2054..2642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2134..2153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2813..3604,3605..3673))
                     /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     3436..3455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3674..3778)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3853..3872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4044..4423
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4424..4627
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4578..4598
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4672..4691
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4672..4690
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(5531..5555)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5563..5586
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(5627..5645)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5628..5645)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5665..5682)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5671..5895
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(5941..6265,1..104)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6028..6047)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6238..6259
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
       61 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
      121 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
      181 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
      241 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
      301 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
      361 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
      421 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
      481 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
      541 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
      601 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
      661 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
      721 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
      781 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
      841 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
      901 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
      961 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     1021 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     1081 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     1141 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     1201 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     1261 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     1321 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     1381 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     1441 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     1501 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     1561 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     1621 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     1681 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     1741 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1801 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1861 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1921 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1981 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2041 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2101 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2161 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2221 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2281 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2341 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2401 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2461 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2521 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2581 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2641 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2701 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2761 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2821 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     2881 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     2941 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3001 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3061 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3121 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3181 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3241 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3301 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3361 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3421 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3481 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3541 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3601 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3661 gttgaatact catactcttc ctttttcatt attattgaag catttatcag ggttattgtc
     3721 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3781 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     3841 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     3901 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     3961 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     4021 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     4081 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     4141 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     4201 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     4261 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     4321 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     4381 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     4441 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     4501 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     4561 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     4621 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac
     4681 tcactatagg gagacccaag cttggtaccg agctcggatc cctacttcca atccaatgcc
     4741 accatgtccc ccttgggagt cctcctgaaa atgatccata atgccctgcg cttcattccc
     4801 gtcacctctg tggtgttgct ttaccaccgc gtccatcctg aggaagtcac cttccacctc
     4861 tacctgatcc caagtgactg ctccattcgg aaggccatag atgatctaga aatgaaattc
     4921 cagtttgtgc gaatccacaa gccacccccg ctgaccccac tttatatggg ctgtcgttac
     4981 actgtgtctg ggtctggttc agggatgctg gaaatactcc ccaaggaact ggagctctgc
     5041 tatcgaagcc ctggagaaga ccagctgttc tcggagttct acgttggcca cttgggatca
     5101 gggatcaggc tgcaagtgaa agacaagaaa gatgagactc tggtgtggga ggccttggtg
     5161 aaaccaggag atctcatgcc tgcaactact ctgatccctc cagcccgcat agccgtacct
     5221 tcacctctgg atgccccgca gttgctgcac tttgtggacc agtatgaaga gcagctgata
     5281 gcccgagtga catcggtgga ggttgtcttg gacaaactgc atggacaggt gctgagccag
     5341 gagcagtacg agagggtgct ggctgagaac acgaggccca gccagatgcg gaagctgttc
     5401 agcttgagcc agtcctggga ccggaagtgc aaagatggac tctaccaagc cctgaaggag
     5461 acccatcctc acctcattat ggaactctgg gagaagggca gcaaaaaggg actcctgcca
     5521 ctcagcagcg acccagcttt cttgtacaaa gtggttgata tcgactacaa ggacgatgac
     5581 gacaagtagt aataacattg gaagtggata atctagaggg ccctattcta tagtgtcacc
     5641 taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     5701 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     5761 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     5821 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     5881 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     5941 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     6001 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     6061 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     6121 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     6181 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     6241 gactcttgtt ccaaactgga acaac
//
