LOCUS       sequence-432151-        6953 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Gateway vector for shuttling NLRP3 C-terminally tagged with 
            mNeonGreen to different destination vectors (start and stop codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6953)
  AUTHORS   Hollingsworth LR, Veeraraghavan P, Paulo JA, Harper JW
  TITLE     Spatiotemporal proteomic profiling of cellular responses to NLRP3 
            agonists
  JOURNAL   bioRxiv 2024.04.19.590338
REFERENCE   2  (bases 1 to 6953)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.04.19.590338"
COMMENT     Sequence Label: pDONR221 hNLRP3-mNG-STOP
FEATURES             Location/Qualifiers
     source          1..6953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(919..946)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1038..1124)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1173..1195
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1188..1204
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1220..1319
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4463..5170
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      4715..4724
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(5172..5271)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5288..5307)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5289..5307)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5312..5328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5312..5328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(5914..6729)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5914..6729)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg ctagccagga
      901 agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc
      961 ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc
     1021 aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
     1081 aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct ggcagttccc
     1141 tactctcgcg ttaacgctag catggatgtt ttcccagtca cgacgttgta aaacgacggc
     1201 cagtcttaag ctcgggcccc aaataatgat tttattttga ctgatagtga cctgttcgtt
     1261 gcaacaaatt gatgagcaat gcttttttat aatgccaact ttgtacaaaa aagcaggctc
     1321 caccatgaag atggcaagca cccgctgcaa gctggccagg tacctggagg acctggagga
     1381 tgtggacttg aagaaattta agatgcactt agaggactat cctccccaga agggctgcat
     1441 ccccctcccg aggggtcaga cagagaaggc agaccatgtg gatctagcca cgctaatgat
     1501 cgacttcaat ggggaggaga aggcgtgggc catggccgtg tggatcttcg ctgcgatcaa
     1561 caggagagac ctttatgaga aagcaaaaag agatgagccg aagtggggtt cagataatgc
     1621 acgtgtttcg aatcccactg tgatatgcca ggaagacagc attgaagagg agtggatggg
     1681 tttactggag tacctttcga gaatctctat ttgtaaaatg aagaaagatt accgtaagaa
     1741 gtacagaaag tacgtgagaa gcagattcca gtgcattgaa gacaggaatg cccgtctggg
     1801 tgagagtgtg agcctcaaca aacgctacac acgactgcgt ctcatcaagg agcaccggag
     1861 ccagcaggag agggagcagg agcttctggc catcggcaag accaagacgt gtgagagccc
     1921 cgtgagtccc attaagatgg agttgctgtt tgaccccgat gatgagcatt ctgagcctgt
     1981 gcacaccgtg gtgttccagg gggcggcagg gattgggaaa acaatcctgg ccaggaagat
     2041 gatgttggac tgggcgtcgg ggacactcta ccaagacagg tttgactatc tgttctatat
     2101 ccactgtcgg gaggtgagcc ttgtgacaca gaggagcctg ggggacctga tcatgagctg
     2161 ctgccccgac ccaaacccac ccatccacaa gatcgtgaga aaaccctcca gaatcctctt
     2221 cctcatggac ggcttcgatg agctgcaagg tgcctttgac gagcacatag gaccgctctg
     2281 cactgactgg cagaaggccg agcggggaga cattctcctg agcagcctca tcagaaagaa
     2341 gctgcttccc gaggcctctc tgctcatcac cacgagacct gtggccctgg agaaactgca
     2401 gcacttgctg gaccatcctc ggcatgtgga gatcctgggt ttctccgagg ccaaaaggaa
     2461 agagtacttc ttcaagtact tctctgatga ggcccaagcc agggcagcct tcagtctgat
     2521 tcaggagaac gaggtcctct tcaccatgtg cttcatcccc ctggtctgct ggatcgtgtg
     2581 cactggactg aaacagcaga tggagagtgg caagagcctt gcccagacat ccaagaccac
     2641 caccgcggtg tacgtcttct tcctttccag tttgctgcag ccccggggag ggagccagga
     2701 gcacggcctc tgcgcccacc tctgggggct ctgctctttg gctgcagatg gaatctggaa
     2761 ccagaaaatc ctgtttgagg agtccgacct caggaatcat ggactgcaga aggcggatgt
     2821 gtctgctttc ctgaggatga acctgttcca aaaggaagtg gactgcgaga agttctacag
     2881 cttcatccac atgactttcc aggagttctt tgccgccatg tactacctgc tggaagagga
     2941 aaaggaagga aggacgaacg ttccagggag tcgtttgaag cttcccagcc gagacgtgac
     3001 agtccttctg gaaaactatg gcaaattcga aaaggggtat ttgatttttg ttgtacgttt
     3061 cctctttggc ctggtaaacc aggagaggac ctcctacttg gagaagaaat taagttgcaa
     3121 gatctctcag caaatcaggc tggagctgct gaaatggatt gaagtgaaag ccaaagctaa
     3181 aaagctgcag atccagccca gccagctgga attgttctac tgtttgtacg agatgcagga
     3241 ggaggacttc gtgcaaaggg ccatggacta tttccccaag attgagatca atctctccac
     3301 cagaatggac cacatggttt cttccttttg cattgagaac tgtcatcggg tggagtcact
     3361 gtccctgggg tttctccata acatgcccaa ggaggaagag gaggaggaaa aggaaggccg
     3421 acaccttgat atggtgcagt gtgtcctccc aagctcctct catgctgcct gttctcatgg
     3481 attggtgaac agccacctca cttccagttt ttgccggggc ctcttttcag ttctgagcac
     3541 cagccagagt ctaactgaat tggacctcag tgacaattct ctgggggacc cagggatgag
     3601 agtgttgtgt gaaacgctcc agcatcctgg ctgtaacatt cggagattgt ggttggggcg
     3661 ctgtggcctc tcgcatgagt gctgcttcga catctccttg gtcctcagca gcaaccagaa
     3721 gctggtggag ctggacctga gtgacaacgc cctcggtgac ttcggaatca gacttctgtg
     3781 tgtgggactg aagcacctgt tgtgcaatct gaagaagctc tggttggtca gctgctgcct
     3841 cacatcagca tgttgtcagg atcttgcatc agtattgagc accagccatt ccctgaccag
     3901 actctatgtg ggggagaatg ccttgggaga ctcaggagtc gcaattttat gtgaaaaagc
     3961 caagaatcca cagtgtaacc tgcagaaact ggggttggtg aattctggcc ttacgtcagt
     4021 ctgttgttca gctttgtcct cggtactcag cactaatcag aatctcacgc acctttacct
     4081 gcgaggcaac actctcggag acaaggggat caaactactc tgtgagggac tcttgcaccc
     4141 cgactgcaag cttcaggtgt tggaattaga caactgcaac ctcacgtcac actgctgctg
     4201 ggatctttcc acacttctga cctccagcca gagcctgcga aagctgagcc tgggcaacaa
     4261 tgacctgggc gacctggggg tcatgatgtt ctgtgaagtg ctgaaacagc agagctgcct
     4321 cctgcagaac ctggggttgt ctgaaatgta tttcaattat gagacaaaaa gtgcgttaga
     4381 aacacttcaa gaagaaaagc ctgagctgac cgtcgtcttt gagccttctt ggttgggcgg
     4441 atccggggga agcggcggat ccgtgagcaa gggcgaggag gataacatgg cctctctccc
     4501 agcgacacat gagttacaca tctttggctc catcaacggt gtggactttg acatggtggg
     4561 tcagggcacc ggcaatccaa atgatggtta tgaggagtta aacctgaagt ccaccaaggg
     4621 tgacctccag ttctccccct ggattctggt ccctcatatc gggtatggct tccatcagta
     4681 cctgccctac cctgacggga tgtcgccttt ccaggccgcc atggtagatg gctccggata
     4741 ccaagtccat cgcacaatgc agtttgaaga tggtgcctcc cttactgtta actaccgcta
     4801 cacctacgag ggaagccaca tcaaaggaga ggcccaggtg aaggggactg gtttccctgc
     4861 tgacggtcct gtgatgacca actcgctgac cgctgcggac tggtgcaggt cgaagaagac
     4921 ttaccccaac gacaaaacca tcatcagtac ctttaagtgg agttacacca ctggaaatgg
     4981 caagcgctac cggagcactg cgcggaccac ctacaccttt gccaagccaa tggcggctaa
     5041 ctatctgaag aaccagccga tgtacgtgtt ccgtaagacg gagctcaagc actccaagac
     5101 cgagctcaac ttcaaggagt ggcaaaaggc ctttaccgat gtgatgggca tggacgagct
     5161 gtacaagtag gacccagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc
     5221 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
     5281 tgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc
     5341 cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     5401 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     5461 aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     5521 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     5581 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     5641 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaccg gcaaacaaac
     5701 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5761 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgacgcgta
     5821 actcacgtta agggattttg gtcatgagct tgcgccgtcc cgtcaagtca gcgtaatgct
     5881 ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg
     5941 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     6001 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     6061 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     6121 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     6181 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     6241 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     6301 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     6361 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     6421 tccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     6481 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     6541 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     6601 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     6661 taaatcagca tccatgttgg aatttaatcg cggcctggag caagacgttt cccgttgaat
     6721 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     6781 tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg
     6841 ttgaataaat cgaacttttg ctgagttgaa ggatcagtca tgaccaaaat cccttaacgt
     6901 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttc
//
