LOCUS       sequence-432147-        6218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway shuttling vector for cloning NLRP3-R262W (no stop codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6218)
  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 6218)
  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: "bioRxiv 
            2024.04.19.590338"
COMMENT     Sequence Label: pDONR221 hNLRP3 R262W (disease-associated muta...
FEATURES             Location/Qualifiers
     source          1..6218
                     /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"
     protein_bind    complement(4437..4536)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4553..4572)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4554..4572)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4577..4593)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4577..4593)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(5179..5994)
                     /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(5179..5994)
                     /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 ccactgttgg 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 ggttggaccc
     4441 agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga
     4501 acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata tcccctatag
     4561 tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct
     4621 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     4681 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     4741 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     4801 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     4861 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     4921 gctgacggaa tttatgcctc ttccgaccat caccggcaaa caaaccaccg ctggtagcgg
     4981 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5041 tttgatcttt tctacggggt ctgacgctca gtggaacgac gcgtaactca cgttaaggga
     5101 ttttggtcat gagcttgcgc cgtcccgtca agtcagcgta atgctctgcc agtgttacaa
     5161 ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     5221 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     5281 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     5341 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     5401 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     5461 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     5521 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     5581 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     5641 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gtttttccgg ggatcgcagt
     5701 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     5761 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     5821 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     5881 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     5941 gttggaattt aatcgcggcc tggagcaaga cgtttcccgt tgaatatggc tcataacacc
     6001 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc
     6061 ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttgttgaa taaatcgaac
     6121 ttttgctgag ttgaaggatc agtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6181 tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
