LOCUS       sequence-324620-        6447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding codon-optimized human NLRP1 
            CARD (dimer interface 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 6447)
  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 6447)
  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 codon-optimized NLRP1 CARD-mCherry...
FEATURES             Location/Qualifiers
     source          1..6447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(120..140)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        125..454
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      305..440
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     367..386
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             521..1315
                     /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(575..594)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1185..1204
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1489..1610
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1526..1545)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1580..1599
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1659..1675)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1659..1675)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1672..1694)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1683..1699
                     /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(1707..1713,1714..1731,1732..1737))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1752..1773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1890..1907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2061..2649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2141..2160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2820..3611,3612..3680))
                     /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     3443..3462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3681..3785)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3860..3879)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4051..4430
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4431..4634
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4585..4605
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4679..4698
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4679..4697
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4745..4754
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5090..5797
                     /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(5232..5250)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5500..5519
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(5816..5834)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5817..5834)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5854..5871)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5860..6084
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6130..6447,1..111)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6217..6236)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(6427..6447,1)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
       61 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
      121 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
      181 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
      241 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
      301 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
      361 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
      421 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
      481 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
      541 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
      601 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
      661 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
      721 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
      781 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
      841 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
      901 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
      961 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     1021 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     1081 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     1141 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     1201 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     1261 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     1321 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     1381 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     1441 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     1501 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     1561 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     1621 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     1681 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     1741 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     1801 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1861 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     1921 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     1981 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2041 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2101 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2161 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2221 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2281 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2341 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2401 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2461 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     2521 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2581 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2641 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     2701 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     2761 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2821 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2881 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2941 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3001 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3061 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3121 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3181 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3241 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3301 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3361 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3421 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3481 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3541 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3601 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3661 cggaaatgtt gaatactcat actcttcctt tttcattatt attgaagcat ttatcagggt
     3721 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3781 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     3841 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     3901 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     3961 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     4021 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     4081 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     4141 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     4201 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     4261 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     4321 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     4381 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     4441 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     4501 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     4561 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     4621 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
     4681 atacgactca ctatagggag acccaagctt ggtaccgagc tcggatccct acttccaatc
     4741 caatgccacc atggacgctc ctcagctgct gcatttcgtg gaccagtacc gggagcagct
     4801 gatcgctaga gtgacctccg tggaagtggt gctggacaag ctgcacggac aggtgctgag
     4861 tcaggagcag tacgagaggg tgctggcaga gaacaccagg cctagccaga tgcggaagct
     4921 gttcagcctg agccagtctt gggacaggaa gtgcaaggac ggcctggccc aggctctgaa
     4981 ggagacacac ccccacctga tcatggagct ctgggagaag ggaagcaaga agggcctgct
     5041 gcctctgtct agcggcattg gtagtgggag caacggcagc agcggatccg tgagcaaggg
     5101 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     5161 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     5221 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     5281 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     5341 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     5401 ggacggcggc gtggtgaccg tgacccagga ctcctccctc caggacggcg agttcatcta
     5461 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     5521 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     5581 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     5641 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     5701 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     5761 ccactccacc ggcggcatgg acgagctgta caagtagtaa tctagagggc cctattctat
     5821 agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     5881 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5941 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     6001 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     6061 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     6121 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     6181 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     6241 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     6301 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     6361 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     6421 ttaatagtgg actcttgttc caaactg
//
