LOCUS       sequence-322628-        5782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding human NLRP1 CARD with a 
            C-terminal FLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5782)
  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 5782)
  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 CARD-FLAG WT
FEATURES             Location/Qualifiers
     source          1..5782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..39
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(158..178)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        163..492
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      343..478
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     405..424
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             559..1353
                     /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(613..632)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1223..1242
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1527..1648
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1564..1583)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1618..1637
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1697..1713)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1697..1713)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1710..1732)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1721..1737
                     /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(1745..1751,1752..1769,1770..1775))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1790..1811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1928..1945)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2099..2687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2179..2198)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2858..3649,3650..3718))
                     /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     3481..3500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3719..3823)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3898..3917)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4089..4468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4469..4672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4623..4643
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4717..4736
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4717..4735
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4783..4792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(5093..5117)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5125..5148
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(5189..5207)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5190..5207)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5227..5244)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5233..5457
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(5503..5782,1..149)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5590..5609)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
       61 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
      121 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
      181 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
      241 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
      301 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
      361 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
      421 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
      481 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
      541 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
      601 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
      661 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
      721 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
      781 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
      841 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
      901 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
      961 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     1021 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     1081 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     1141 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     1201 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     1261 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     1321 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     1381 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     1441 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     1501 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     1561 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     1621 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     1681 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1741 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1801 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1861 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1921 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1981 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2041 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2101 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2161 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2221 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2281 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2341 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2401 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2461 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2521 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2581 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2641 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2701 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2761 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2821 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2881 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2941 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3001 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3061 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3121 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3181 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3241 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3301 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3361 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3421 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3481 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3541 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3601 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3661 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3721 tcttcctttt tcattattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3781 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3841 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     3901 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     3961 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     4021 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     4081 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     4141 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     4201 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     4261 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     4321 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     4381 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     4441 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     4501 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     4561 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     4621 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct
     4681 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
     4741 ccaagcttgg taccgagctc ggatccctac ttccaatcca atgccaccat ggatgccccg
     4801 cagttgctgc actttgtgga ccagtatcga gagcagctga tagcccgagt gacatcggtg
     4861 gaggttgtct tggacaaact gcatggacag gtgctgagcc aggagcagta cgagagggtg
     4921 ctggctgaga acacgaggcc cagccagatg cggaagctgt tcagcttgag ccagtcctgg
     4981 gaccggaagt gcaaagatgg actctaccaa gccctgaagg agacccatcc tcacctcatt
     5041 atggaactct gggagaaggg cagcaaaaag ggactcctgc cactcagcag cgacccagct
     5101 ttcttgtaca aagtggttga tatcgactac aaggacgatg acgacaagta gtaataacat
     5161 tggaagtgga taatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct
     5221 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     5281 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     5341 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     5401 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     5461 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     5521 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5581 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5641 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5701 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5761 tttttcgccc tttgacgttg ga
//
