LOCUS       Exported                8537 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses N-terminal Flag-tagged NLRP3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3-N-Flag-NLRP3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8537)
  AUTHORS   Shi H, Wang Y, Li X, Zhan X, Tang M, Fina M, Su L, Pratt D, Bu CH, 
            Hildebrand S, Lyon S, Scott L, Quan J, Sun Q, Russell J, Arnett S, 
            Jurek P, Chen D, Kravchenko VV, Mathison JC, Moresco EM, Monson NL, 
            Ulevitch RJ, Beutler B
  TITLE     NLRP3 activation and mitosis are mutually exclusive events 
            coordinated by NEK7, a new inflammasome component.
  JOURNAL   Nat Immunol. 2016 Mar;17(3):250-8. doi: 10.1038/ni.3333. Epub 2015 
            Dec 7.
  PUBMED    26642356
REFERENCE   2  (bases 1 to 8537)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8537
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             922..945
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(4090..4108)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4091..4108)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4128..4145)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4134..4358
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4404..4832
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4491..4510)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4701..4722
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4841..4861)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4846..5175
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5026..5161
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5088..5107
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5242..6036
                     /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(5296..5315)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5906..5925
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6210..6331
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6247..6266)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6301..6320
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6380..6396)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6380..6396)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6393..6415)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6404..6420
                     /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(6428..6458)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6473..6494
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6611..6628)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6782..7370)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6862..6881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7541..8401)
                     /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     8164..8183
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8402..8506)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgcccaccat ggactacaaa gacgatgacg ataaaatcga tatcgaattc
      961 atgacgagtg tccgttgcaa gctggctcag tatctagagg accttgaaga tgtggacctc
     1021 aagaaattca aaatgcattt ggaagattac ccgcccgaga aaggctgtat cccagtcccc
     1081 aggggccaga tggagaaggc agatcacttg gatctagcca cactcatgat tgacttcaat
     1141 ggtgaggaga aggcctgggc catggctgtg tggatctttg ctgcgatcaa caggcgagac
     1201 ctctgggaaa aagctaagaa ggaccagcca gagtggaatg acacgtgtac atcacattcc
     1261 tctatggtat gccaggagga cagccttgaa gaagagtgga tgggtttgct gggatatctc
     1321 tcccgcatct ccatttgtaa aaagaagaaa gattactgta agatgtacag acgacatgtg
     1381 agaagcaggt tctactctat caaggacagg aacgcgcgtc taggtgagag tgtggacctc
     1441 aacagtcgct acacgcagct ccaactggtc aaggagcatc caagcaagca ggagcgggag
     1501 catgaactcc tgaccatcgg ccggactaaa atgcgggaca gccccatgag ttcccttaag
     1561 ctggagctgc tgtttgagcc cgaggacggg cactcggagc ctgtgcacac agtggtgttc
     1621 cagggagcag caggcatcgg gaaaaccatc ctagccagga agattatgtt ggactgggca
     1681 ctgggaaagc tcttcaaaga caaatttgac tatttgttct ttatccactg ccgagaggtg
     1741 agcctcagga cgccaaggag tctagcagac ctgattgtca gctgctggcc tgacccaaac
     1801 ccaccagtgt gcaagatcct gcgcaagcct tccaggatcc tcttcctcat ggatggcttt
     1861 gatgagctac aaggggcctt tgacgagcac attggggagg tctgcacaga ctggcaaaag
     1921 gctgtgcggg gagacattct gctaagcagc ctcatccgaa agaaactgct gcccaaggcc
     1981 tctctgctca taacgacgag gccggtagcc ttggagaaac tgcagcatct cctggaccac
     2041 ccccgccatg tggagatcct aggtttctct gaggccaaaa ggaaggagta tttctttaag
     2101 tatttctcca acgagctgca ggcccgggag gccttcaggc tgatccaaga gaatgaggtc
     2161 ctctttacca tgtgcttcat ccccctggtc tgctggattg tgtgcacggg gctaaagcaa
     2221 cagatggaga ccgggaagag cctggcccag acctccaaga ccactacggc cgtctacgtc
     2281 ttcttccttt ccagcctgct gcaatcccgg gggggcattg aggagcatct cttctctgac
     2341 tacctacagg ggctctgttc actggctgcg gatggaattt ggaaccagaa aatcctattt
     2401 gaggagtgtg atctgcggaa gcacggcctg cagaagactg acgtctccgc tttcctgagg
     2461 atgaacgtgt tccagaagga agtggactgc gagagattct acagcttcag ccacatgact
     2521 ttccaggagt tcttcgctgc tatgtactat ttgctggaag aggaggcaga gggggagacc
     2581 gtgaggaaag gaccaggagg ttgttcagat cttctgaacc gagacgtgaa ggtcctacta
     2641 gaaaattacg gcaagtttga aaaaggctat ctgatttttg ttgtccgatt cctctttggc
     2701 cttgtaaacc aggagagaac ctcttatttg gagaagaaac taagttgcaa gatctctcag
     2761 caagtcagac tggaactact gaagtggatt gaagtgaaag ccaaggccaa gaagctgcag
     2821 tggcagccca gccaactgga actgttctac tgcctgtacg agatgcagga ggaagacttt
     2881 gtgcagagtg ccatggacca ctttcccaaa attgagatca acctctctac cagaatggac
     2941 cacgtggttt cctccttttg tattaagaac tgtcataggg tcaaaacgct ttccctgggt
     3001 ttttttcaca actcgcccaa ggaggaagaa gaagagagga gaggaggtcg acccttggac
     3061 caggttcagt gtgttttccc agacactcat gttgcctgtt cttccagact ggtgaactgc
     3121 tgcctcactt ctagcttctg ccgtggtctc ttctcaagtc taagcaccaa ccggagcctc
     3181 actgaactgg acctcagtga caatactctg ggagacccgg gcatgagggt gctgtgtgag
     3241 gcactccagc acccaggctg taacattcag agactgtggt tggggcgctg cggactgtcc
     3301 catcaatgct gcttcgacat ctcctctgtc ctgagcagca gccagaagct ggtggagctg
     3361 gacctcagtg acaatgccct gggggacttt ggaatcagat tgctgtgtgt gggactgaag
     3421 cacctgctct gcaacctcca gaaactgtgg ttggtgagct gctgtctcac atccgcgtgt
     3481 tgtcaggatc tcgcattggt tctgagctcc aaccattctc tgaccagact gtacattgga
     3541 gaaaatgcct tgggagactc aggagtccaa gttttgtgtg aaaagatgaa ggacccacag
     3601 tgtaacttgc agaagctggg gttggtgaat tccggcctta cttcaatctg ttgttcagct
     3661 ctgacctctg tgctcaaaac caaccagaac ttcacacacc tctatctacg aagcaatgcc
     3721 cttggagaca caggactcag gctcctctgt gaggggcttc tgcacccgga ctgtaaacta
     3781 cagatgctgg aattagacaa ctgcagcctc acctcacaca gctgctggaa tctctccaca
     3841 attctgaccc acaaccacag ccttcggaag ctgaacctgg gcaacaatga tcttggcgat
     3901 ctgtgcgtgg tgaccctctg tgaggtgctg aaacagcagg gctgcctcct gcagagccta
     3961 cagttgggtg aaatgtactt aaatcgtgaa acaaaacgtg ccttagaagc gctccaggaa
     4021 gaaaagcctg agctgactat agtcttcgag atttcctggt agctcgagca tgcatctaga
     4081 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     4141 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4201 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4261 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4321 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     4381 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4441 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4501 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4561 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4621 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4681 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4741 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4801 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     4861 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4921 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4981 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     5041 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     5101 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     5161 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     5221 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     5281 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5341 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5401 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     5461 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5521 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5581 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5641 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5701 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5761 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5821 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5881 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5941 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     6001 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     6061 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     6121 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     6181 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     6241 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     6301 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     6361 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     6421 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     6481 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6541 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     6601 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     6661 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6721 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6781 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6841 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6901 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6961 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     7021 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     7081 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     7141 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     7201 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     7261 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     7321 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     7381 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     7441 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     7501 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     7561 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     7621 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     7681 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7741 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     7801 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7861 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7921 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7981 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     8041 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     8101 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     8161 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     8221 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     8281 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     8341 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     8401 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     8461 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     8521 aagtgccacc tgacgtc
//
