LOCUS       sequence-324554-        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 E1411R
FEATURES             Location/Qualifiers
     source          1..6265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      179..314
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     241..260
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             395..1189
                     /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(449..468)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1059..1078
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1363..1484
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1400..1419)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1454..1473
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1533..1549)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1533..1549)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1546..1568)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1557..1573
                     /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(1581..1587,1588..1605,1606..1611))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1626..1647
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1764..1781)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1935..2523)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2015..2034)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2694..3485,3486..3554))
                     /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     3317..3336
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3555..3659)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3734..3753)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3925..4304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4305..4508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4459..4479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4553..4572
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4553..4571
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(5412..5436)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5444..5467
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(5508..5526)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5509..5526)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5546..5563)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5552..5776
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5822..6250
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5909..5928)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6119..6140
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(join(6259..6265,1..14))
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6264..6265,1..328)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
       61 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
      121 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
      181 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
      241 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
      301 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
      361 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
      421 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
      481 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
      541 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
      601 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
      661 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
      721 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
      781 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
      841 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
      901 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
      961 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     1021 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     1081 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     1141 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     1201 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     1261 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     1321 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     1381 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     1441 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     1501 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     1561 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     1621 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     1681 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1741 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     1801 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1861 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1921 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     1981 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2041 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2101 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     2161 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2221 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2281 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2341 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     2401 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     2461 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     2521 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     2581 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2641 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2701 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2761 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2821 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2881 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2941 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3001 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3061 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3121 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3181 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3241 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3301 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3361 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3421 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3481 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3541 tgttgaatac tcatactctt cctttttcat tattattgaa gcatttatca gggttattgt
     3601 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     3661 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     3721 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     3781 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     3841 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     3901 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
     3961 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     4021 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     4081 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     4141 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     4201 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     4261 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     4321 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     4381 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     4441 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     4501 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
     4561 ctcactatag ggagacccaa gcttggtacc gagctcggat ccctacttcc aatccaatgc
     4621 caccatgtcc cccttgggag tcctcctgaa aatgatccat aatgccctgc gcttcattcc
     4681 cgtcacctct gtggtgttgc tttaccaccg cgtccatcct gaggaagtca ccttccacct
     4741 ctacctgatc ccaagtgact gctccattcg gaaggccata gatgatctag aaatgaaatt
     4801 ccagtttgtg cgaatccaca agccaccccc gctgacccca ctttatatgg gctgtcgtta
     4861 cactgtgtct gggtctggtt cagggatgct ggaaatactc cccaaggaac tggagctctg
     4921 ctatcgaagc cctggagaag accagctgtt ctcggagttc tacgttggcc acttgggatc
     4981 agggatcagg ctgcaagtga aagacaagaa agatgagact ctggtgtggg aggccttggt
     5041 gaaaccagga gatctcatgc ctgcaactac tctgatccct ccagcccgca tagccgtacc
     5101 ttcacctctg gatgccccgc agttgctgca ctttgtggac cagtatcgag agcagctgat
     5161 agcccgagtg acatcggtgg aggttgtctt ggacaaactg catggacagg tgctgagcca
     5221 gaggcagtac gagagggtgc tggctgagaa cacgaggccc agccagatgc ggaagctgtt
     5281 cagcttgagc cagtcctggg accggaagtg caaagatgga ctctaccaag ccctgaagga
     5341 gacccatcct cacctcatta tggaactctg ggagaagggc agcaaaaagg gactcctgcc
     5401 actcagcagc gacccagctt tcttgtacaa agtggttgat atcgactaca aggacgatga
     5461 cgacaagtag taataacatt ggaagtggat aatctagagg gccctattct atagtgtcac
     5521 ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5581 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5641 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5701 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     5761 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     5821 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     5881 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5941 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     6001 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     6061 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     6121 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     6181 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     6241 aacgcgaatt aattctgtgg aatgt
//
