LOCUS       sequence-324560-        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 E1414R
FEATURES             Location/Qualifiers
     source          1..6265
                     /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"
     protein_bind    complement(1488..1512)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1520..1543
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1584..1602)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1585..1602)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1622..1639)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1628..1852
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1898..2326
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1985..2004)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2195..2216
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2335..2355)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2340..2669
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2520..2655
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2582..2601
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2736..3530
                     /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(2790..2809)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3400..3419
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3704..3825
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3741..3760)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3795..3814
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3874..3890)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3874..3890)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3887..3909)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3898..3914
                     /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(3922..3928,3929..3946,3947..3952))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3967..3988
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4105..4122)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4276..4864)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4356..4375)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5035..5826,5827..5895))
                     /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     5658..5677
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5896..6000)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6075..6094)
                     /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 atgtccccct tgggagtcct
      721 cctgaaaatg atccataatg ccctgcgctt cattcccgtc acctctgtgg tgttgcttta
      781 ccaccgcgtc catcctgagg aagtcacctt ccacctctac ctgatcccaa gtgactgctc
      841 cattcggaag gccatagatg atctagaaat gaaattccag tttgtgcgaa tccacaagcc
      901 acccccgctg accccacttt atatgggctg tcgttacact gtgtctgggt ctggttcagg
      961 gatgctggaa atactcccca aggaactgga gctctgctat cgaagccctg gagaagacca
     1021 gctgttctcg gagttctacg ttggccactt gggatcaggg atcaggctgc aagtgaaaga
     1081 caagaaagat gagactctgg tgtgggaggc cttggtgaaa ccaggagatc tcatgcctgc
     1141 aactactctg atccctccag cccgcatagc cgtaccttca cctctggatg ccccgcagtt
     1201 gctgcacttt gtggaccagt atcgagagca gctgatagcc cgagtgacat cggtggaggt
     1261 tgtcttggac aaactgcatg gacaggtgct gagccaggag cagtaccgga gggtgctggc
     1321 tgagaacacg aggcccagcc agatgcggaa gctgttcagc ttgagccagt cctgggaccg
     1381 gaagtgcaaa gatggactct accaagccct gaaggagacc catcctcacc tcattatgga
     1441 actctgggag aagggcagca aaaagggact cctgccactc agcagcgacc cagctttctt
     1501 gtacaaagtg gttgatatcg actacaagga cgatgacgac aagtagtaat aacattggaa
     1561 gtggataatc tagagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca
     1621 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1681 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1741 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     1801 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     1861 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     1921 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1981 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2041 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2101 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2161 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2221 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2281 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     2341 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2401 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2461 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     2521 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     2581 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     2641 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     2701 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     2761 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     2821 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     2881 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     2941 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3001 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3061 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3121 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3181 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     3241 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     3301 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     3361 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     3421 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     3481 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     3541 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     3601 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     3661 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     3721 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3781 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     3841 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3901 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     3961 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4021 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4081 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4141 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4201 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4261 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4321 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4381 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4441 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4501 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4561 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4621 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4681 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4741 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4801 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4861 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4921 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4981 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5041 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5101 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5161 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5221 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5281 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5341 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5401 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5461 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5521 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5581 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5641 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5701 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5761 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5821 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5881 atgttgaata ctcatactct tcctttttca ttattattga agcatttatc agggttattg
     5941 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6001 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     6061 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     6121 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     6181 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     6241 gatgtacggg ccagatatac gcgtt
//
