LOCUS       sequence-496400-        7524 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Expresses human TOP3B (Y336F; catalytically inactive mutant) with a 
            C-terminal 3xFLAG tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7524)
  AUTHORS   Warrick JE, Attili D, van Eeuwen T, Pastore B, Hoffmann-Weitsman SE,
            Forsyth NC, Tang W, Barmada SJ, Kearse MG
  TITLE     An autism spectrum disorder mutation in Topoisomerase 3beta causes 
            accumulation of covalent mRNA intermediates by disrupting metal 
            binding within the zinc finger domain.
  JOURNAL   Nucleic Acids Res. 2025 Oct 28;53(20):gkaf1138. doi: 
            10.1093/nar/gkaf1138.
  PUBMED    41189056
REFERENCE   2  (bases 1 to 7524)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 7, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "2025-10-28"; volume: "53(20):gkaf1138. doi"; 
            pages: " 10.1093/nar/gkaf1138"
COMMENT     Sequence Label: pCMV6-TOP3B (Y336F)-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..173
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     165..182
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     166..182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        343..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        723..926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     877..897
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     952..971
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        952..970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1448..1457
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3642..3671
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3690..3755
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3775..3791)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3775..3791)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3808..4430
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4007..4026)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(4579..5167)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4659..4678)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5496..5543
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     5568..5587
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(5775..6569)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(5775..6569)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5886..5905)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6496..6515
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6604..6961)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6618..6753
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6770..6961
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6918..6938
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(6963..7067)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7094..7522
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7181..7200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7391..7412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
       61 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      121 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      181 gtgccaagct gatctataca ttgaatcaat attggcaatt agccatatta gtcattggtt
      241 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata
      301 tgtacattta tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta
      361 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      421 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      481 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      541 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      601 gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      661 tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      721 tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat
      781 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      841 actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac
      901 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagaatttt gtaatacgac
      961 tcactatagg gcggccggga attcgtcgac tggatccggt accgaggaga tctgccgccg
     1021 cgatcgccat gaagactgtg ctcatggttg ctgaaaagcc gtccttggca cagtcaattg
     1081 ccaaaatcct ctctagaggg agcctgtcct cacacaaagg gctgaacggg gcctgctcag
     1141 tccacgagta cactgggacc tttgctggcc agccagtgcg cttcaagatg acgtctgtct
     1201 gtggtcacgt gatgaccctg gatttcctgg gaaaatacaa caaatgggac aaagtggacc
     1261 ccgcagaact gttcagccaa gctcccacgg agaagaaaga agctaacccc aagctgaaca
     1321 tggtgaagtt cctgcaggtg gagggcagag gctgcgacta catcgtgctg tggctggact
     1381 gcgacaagga gggggagaac atctgctttg aggttcttga tgctgttctg cccgtcatga
     1441 acaaggccca tggtggcgag aagaccgtgt tccgggccag gtttagctcc atcacggaca
     1501 cagacatctg taatgccatg gcctgcctag gcgagcctga ccacaacgag gcgctctcag
     1561 tggatgctcg ccaggagctg gacctgcgaa tcggctgtgc attcaccagg tttcagacta
     1621 aatatttcca ggggaaatac ggtgatttag acagctctct catctccttt gggccgtgtc
     1681 agactccaac cctgggattc tgtgtggaga gacatgataa aatccagtcc ttcaaaccag
     1741 agacctactg ggtgctgcag gccaaggtta acactgacaa agacagatct ctccttttgg
     1801 actgggaccg agtaagagtg tttgaccggg agatcgcaca gatgttttta aacatgacaa
     1861 agctggagaa ggaagcccag gtggaggcca caagcaggaa agaaaaggcc aagcagaggc
     1921 ccctggccct gaacactgtg gagatgctgc gtgtggccag ctcttctctg ggcatggggc
     1981 cgcagcacgc catgcagacg gctgagcggc tctacacgca aggctacatc agctttccac
     2041 ggacagagac cacccactac cctgagaact ttgacctgaa gggctctctg cggcagcagg
     2101 ccaaccaccc ctactgggcc gacacggtga agcggttgtt agcagaaggt atcaaccgcc
     2161 cgcggaaagg ccatgacgcc ggcgaccatc cccccatcac ccccatgaag tctgccacag
     2221 aggccgaatt agggggtgac gcgtggcggc tctatgagta catcaccaga cacttcatcg
     2281 ccacggtcag ccatgactgc aagtacctgc agagcaccat ctccttcaga attgggcccg
     2341 agctcttcac ctgctccggg aagaccgtcc tctcaccagg cttcacggag gtcatgccct
     2401 ggcagagcgt gcccctggag gagagcctgc ccacttgcca gcggggtgat gccttccctg
     2461 tgggcgaggt gaagatgctg gagaagcaga cgaacccacc cgactacctg acggaggccg
     2521 agctcatcac gctcatggag aagcatggca tcggcacgga tgccagcatc cctgtgcata
     2581 tcaacaacat ctgccagcgc aactatgtca cggtggagag cgggcgccgg ctcaagccca
     2641 ccaacctcgg catcgtcctg gtgcacggct actataagat tgatgcagag ctggtgctcc
     2701 ccaccatccg cagtgcagtg gagaagcagc tgaacctgat cgcccagggc aaggccgact
     2761 accgccaggt cctgggccac accctggacg tgttcaagag gaagttccac tactttgtcg
     2821 actccattgc tggcatggat gagttgatgg aggtgtcttt ctcgcccctg gcggccacag
     2881 gcaagcccct ctcacgctgt gggaagtgcc accgcttcat gaagtacatc caggccaagc
     2941 caagccgcct gcactgctcc cactgcgatg agacctacac gctcccccag aacggcacca
     3001 tcaagctcta caaggagctc cgctgccctc tggatgactt cgagctggtc ctgtggtcat
     3061 caggctctcg gggcaagagc tacccgctgt gcccctactg ctacaaccac ccacccttcc
     3121 gagacatgaa gaaaggcatg ggctgcaacg agtgtacgca cccctcctgc cagcactcgc
     3181 tgagcatgct gggcatcggc cagtgcgtgg aatgtgagag cggggtgctg gtgctggacc
     3241 ccacctcggg ccccaagtgg aaggtggcct gcaacaagtg caacgtggta gcgcactgct
     3301 tcgagaacgc ccaccgcgtg cgggtgtccg ccgacacctg cagtgtctgt gaggccgcct
     3361 tgcttgatgt ggacttcaac aaggccaagt ccccactccc gggcgatgag acgcagcaca
     3421 tgggctgcgt cttttgtgac cccgtcttcc aggagctggt ggagctgaag catgcggcct
     3481 cctgccaccc catgcaccgc ggtggaccag ggagaaggca gggtcgaggg cggggccggg
     3541 ccaggaggcc ccctgggaag cccaacccca gacggcccaa ggacaagatg tcagccctgg
     3601 ccgcctactt tgtaagcgga ccgacgcgta cgcggccgct cgagcagaaa ctcatctcag
     3661 aagaggatct ggcagcaaat gatatcctgg actacaaaga ccatgacggt gattataaag
     3721 atcatgacat cgattacaag gatgacgatg acaaggttta aacggccggc cgcggtcata
     3781 gctgtttcct gaagcttaac agatcccggg tggcatccct gtgacccctc cccagtgcct
     3841 ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt
     3901 tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta
     3961 tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca
     4021 agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg
     4081 attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc
     4141 taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac
     4201 tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga
     4261 accactgctc ccttccctgt ccttctgatt ttaaaataac tataccagca ggaggacgtc
     4321 cagacacagc ataggctacc tggccatgcc caaccggtgg gacatttgag ttgcttgctt
     4381 ggcactgtcc tctcatgcgt tgggtccact cagtagatgc ctgttgaatt gggtacgcgg
     4441 ccagcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4501 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4561 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4621 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4681 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4741 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4801 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4861 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4921 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4981 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5041 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5101 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5161 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5221 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5281 ttaaaaatga agttttaaat caatctaaag tatatatgag taacctgagg ctatggcagg
     5341 gcctgccgcc ccgacgttgg ctgcgagccc tgggccttca cccgaacttg gggggtgggg
     5401 tggggaaaag gaagaaacgc gggcgtattg gccccaatgg ggtctcggtg gggtatcgac
     5461 agagtgccag ccctgggacc gaaccccgcg tttatgaaca aacgacccaa caccgtgcgt
     5521 tttattctgt ctttttattg ccgtcatagc gcgggttcct tccggtattg tctccttccg
     5581 tgtttcagtt agcctccccc tagggtgggc gaagaactcc agcatgagat ccccgcgctg
     5641 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     5701 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     5761 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     5821 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     5881 tcagcaatat cacgggtagc caacgctatg tcctgatagc gatccgccac acccagccgg
     5941 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     6001 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc tcgccttgag cctggcgaac
     6061 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     6121 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     6181 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     6241 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     6301 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     6361 agccacgata gccgcgctgc ctcgtcttgc agttcattca gggcaccgga caggtcggtc
     6421 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     6481 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     6541 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatcg
     6601 atctttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga
     6661 ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga
     6721 atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt
     6781 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt
     6841 tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     6901 gcctggggac tttccacacc ctaactgaca cacattccac agctggttct ttccgcctca
     6961 ggactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7021 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7081 aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     7141 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     7201 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     7261 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     7321 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     7381 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     7441 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     7501 taacaaaaat ttaacgcgaa tttt
//
