LOCUS       sequence-201400-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of TDP-43 S48E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  AUTHORS   Wang A, Conicella AE, Schmidt HB, Martin EW, Rhoads SN, Reeb AN, 
            Nourse A, Ramirez Montero D, Ryan VH, Rohatgi R, Shewmaker F, Naik 
            MT, Mittag T, Ayala YM, Fawzi NL
  TITLE     A single N-terminal phosphomimic disrupts TDP-43 polymerization, 
            phase separation, and RNA splicing.
  JOURNAL   EMBO J. 2018 Mar 1;37(5). pii: embj.201797452. doi: 
            10.15252/embj.201797452. Epub 2018 Feb 9.
  PUBMED    29438978
REFERENCE   2  (bases 1 to 6390)
  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: "EMBO J."; 
            date: "2018-03-1"; volume: "37(5). pii: embj.201797452. doi"; pages:
            " 10.15252/embj.201797452. Epub 2018 Feb 9"
COMMENT     Sequence Label: pJ4M/TDP-43 S48E
FEATURES             Location/Qualifiers
     source          1..6390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     CDS             1341..1361
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1371..2468
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2325..2334
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2442..2465
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    2487..2511
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2520..2537
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2545..2592
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2815..2834)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2901..2992
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2993..3802
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3885..4967
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3904..3923)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      5053..5641
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5542..5561
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5795..5812
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5910..5927
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5911..5927
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt ttgaaggaga tatacccata tgatgagcga gtatattcgt gttaccgaag
      121 atgagaacga cgagccgatc gagattccga gcgaagatga cggtacggtc ttgctgagca
      181 cggtgaccgc gcagttccca ggcgcctgtg gcctgcgcta tcgtaatccg gtcgagcagt
      241 gcatgcgcgg tgtccgtctg gttgagggca ttctgcacgc tccggacgcg ggctggggca
      301 atctggtgta cgtggtcaat tacccgaaag acaacaagcg caagatggat gaaactgacg
      361 ccagctctgc agttaaagtc aagcgtgcag tgcagaaaac gagcgacctg atcgtgctgg
      421 gtttgccgtg gaaaaccacg gagcaagacc tgaaagaata ctttagcacc ttcggtgagg
      481 tgctgatggt ccaggtaaag aaagatctga aaaccggtca cagcaagggt tttggcttcg
      541 tccgtttcac cgagtatgaa acccaggtga aagttatgtc gcaacgtcac atgatcgacg
      601 gtcgttggtg tgactgcaag ctgccgaaca gcaagcagag ccaagatgaa ccgctgcgct
      661 cccgtaaagt tttcgttggt cgctgcaccg aagatatgac ggaagatgag ctgcgtgagt
      721 tcttttctca atatggtgac gtgatggatg ttttcatccc gaaacctttc cgcgcctttg
      781 catttgttac cttcgctgac gatcaaatcg cgcagagcct gtgtggtgag gatctgatta
      841 tcaaaggtat ttccgttcac atttctaacg cggagccaaa gcataacagc aaccgccaac
      901 tggaacgttc cggtcgtttc ggtggtaatc cgggtggttt cggcaaccag ggtggctttg
      961 gcaattcgcg tggcggtggt gccggcttgg gcaataacca aggttccaat atgggtggcg
     1021 gtatgaactt tggtgccttc tccatcaatc cggcaatgat ggcggctgca caagcggcgc
     1081 tgcagtctag ctggggtatg atgggcatgc tggcgagcca gcagaaccag tcaggcccga
     1141 gcggcaataa tcagaatcaa ggtaacatgc agcgtgaacc gaatcaagcg tttggtagcg
     1201 gtaacaatag ctacagcggt agcaacagcg gtgcggcaat cggctggggt agcgcaagca
     1261 atgcgggcag cggtagcggt ttcaacggcg gctttggtag cagcatggat agcaagagct
     1321 ccggttgggg catgctcgag gagaacctgt actttcaggg tagcggtacc aaaatcgaag
     1381 aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg
     1441 gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg
     1501 aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac
     1561 acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag
     1621 cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga
     1681 ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga
     1741 acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta
     1801 agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg
     1861 acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg
     1921 ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca
     1981 tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga
     2041 tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg
     2101 taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg
     2161 caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc
     2221 tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc
     2281 tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccacc atggaaaacg
     2341 cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc
     2401 gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg
     2461 cgcagactaa ttcgggatct gatatcacaa gtttgtacaa aaaagctgaa ggcggtaccc
     2521 atcaccatca ccatcattga ccccctagca taaccccttg gggcctctaa acgggtcttg
     2581 aggggttttt tgcccctgag acgcgtcaat cgagttcgta cctaagggcg acacccccta
     2641 attagcccgg gcgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgg
     2701 cagttcccta ctctcgcatg gggagtcccc acactaccat cggcgctacg gcgtttcact
     2761 tctgagttcg gcatggggtc aggtgggacc accgcgctac tgccgccagg caaacaaggg
     2821 gtgttatgag ccatattcag gtataaatgg gctcgcgata atgttcagaa ttggttaatt
     2881 ggttgtaaca ctgaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2941 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga atatgagcca
     3001 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     3061 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     3121 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     3181 tacagatgag atggtcagac taaactggct gacggaattt atgccacttc cgaccatcaa
     3241 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     3301 agcgttccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     3361 agtgttcctg cgccggttgc actcgattcc tgtttgtaat tgtcctttta acagcgatcg
     3421 cgtatttcgc ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     3481 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     3541 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     3601 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     3661 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     3721 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc aatttcattt
     3781 gatgctcgat gagtttttct aagcggcgcg ccatcgaatg gcgcaaaacc tttcgcggta
     3841 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatatgaaa ccagtaacgt
     3901 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     3961 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4021 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4081 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4141 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     4201 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     4261 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     4321 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaggacg
     4381 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     4441 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     4501 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     4561 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     4621 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     4681 cggatatctc ggtagtggga tacgacgata ccgaagatag ctcatgttat atcccgccgt
     4741 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     4801 aactctctca gggccaggcg gtgaagggca atcagctgtt gccagtctca ctggtgaaaa
     4861 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     4921 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgactc atgaccaaaa
     4981 tcccttaacg tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     5041 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5101 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5161 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5221 agcccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5281 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5341 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5401 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5461 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5521 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5581 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5641 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5701 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5761 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5821 aggcgagagt agggaactgc caggcatcaa actaagcaga aggcccctga cggatggcct
     5881 ttttgcgttt ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt aagctcgggc
     5941 cccctgggcg gttctgataa cgagtaatcg ttaatccgca aataacgtaa aaacccgctt
     6001 cggcgggttt ttttatgggg ggagtttagg gaaagagcat ttgtcagaat atttaagggc
     6061 gcctgtcact ttgcttgata tatgagaatt atttaacctt ataaatgaga aaaaagcaac
     6121 gcactttaaa taagatacgt tgctttttcg attgatgaac acctataatt aaactattca
     6181 tctattattt atgatttttt gtatatacaa tatttctagt ttgttaaaga gaattaagaa
     6241 aataaatctc gaaaataata aagggaaaat cagtttttga tatcaaaatt atacatgtca
     6301 acgataatac aaaatataat acaaactata agatgttatc agtatttatt atgcatttag
     6361 aataaatttt gtgtcgccct tccgcgaaat
//
