LOCUS       sequence-345524-        7286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7286)
  AUTHORS   Johnson BS, Snead D, Lee JJ, McCaffery JM, Shorter J, Gitler AD
  TITLE     TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral 
            sclerosis-linked mutations accelerate aggregation and increase 
            toxicity.
  JOURNAL   J Biol Chem. 2009 Jul 24. 284(30):20329-39.
  PUBMED    19465477
REFERENCE   2  (bases 1 to 7286)
  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: "J Biol 
            Chem. 2009 Jul 24. 284(30):20329-39."
COMMENT     Sequence Label: pDuet TDP-43 Q331K
FEATURES             Location/Qualifiers
     source          1..7286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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)."
     RBS             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             69..725
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     683..705
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             741..761
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        2081..2099
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2100..2124
                     /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             2233..2277
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2315..2333)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2329..2376
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2413..2868
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2500..2519)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2710..2731
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2983..3774,3775..3843))
                     /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     3606..3625
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3844..3936)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4017..4605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4506..4525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4759..4776
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4791..4930
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4856..4875)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5016..5038
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5032..5223)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5795..6877)
                     /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     6839..6858
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6878..6955)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7161..7180
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7268..7286
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggctcccct atactaggtt attggaaaat taagggcctt gtgcaaccca
      121 ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg
      181 aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt
      241 attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg
      301 acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag
      361 gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa
      421 ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt
      481 tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg
      541 acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag
      601 tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca
      661 agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc
      721 caaaatcgga tctggctagc gagaatcttt attttcaggg atccatgtct gaatatattc
      781 gggtaaccga agatgagaac gatgagccca ttgaaatacc atcggaagac gatgggacgg
      841 tgctgctctc cacggttaca gcccagtttc caggggcgtg tgggcttcgc tacaggaatc
      901 cagtgtctca gtgtatgaga ggtgtccggc tggtagaagg aattctgcat gccccagatg
      961 ctggctgggg aaatctggtg tatgttgtca actatccaaa agataacaaa agaaaaatgg
     1021 atgagacaga tgcttcatca gcagtgaaag tgaaaagagc agtccagaaa acatccgatt
     1081 taatagtgtt gggtctccca tggaaaacaa ccgaacagga cctgaaagag tattttagta
     1141 cctttggaga agttcttatg gtgcaggtca agaaagatct taagactggt cattcaaagg
     1201 ggtttggctt tgttcgtttt acggaatatg aaacacaagt gaaagtaatg tcacagcgac
     1261 atatgataga tggacgatgg tgtgactgca aacttcctaa ttctaagcaa agccaagatg
     1321 agcctttgag aagcagaaaa gtgtttgtgg ggcgctgtac agaggacatg actgaggatg
     1381 agctgcggga gttcttctct cagtacgggg atgtgatgga tgtcttcatc cccaagccat
     1441 tcagggcctt tgcctttgtt acatttgcag atgatcagat tgcgcagtct ctttgtggag
     1501 aggacttgat cattaaagga atcagcgttc atatatccaa tgccgaacct aagcacaata
     1561 gcaatagaca gttagaaaga agtggaagat ttggtggtaa tccaggtggc tttgggaatc
     1621 agggtggatt tggtaatagc agagggggtg gagctggttt gggaaacaat caaggtagta
     1681 atatgggtgg tgggatgaac tttggtgcgt tcagcattaa tccagccatg atggctgccg
     1741 cccaggcagc actaaagagc agttggggta tgatgggcat gttagccagc cagcagaacc
     1801 agtcaggccc atcgggtaat aaccaaaacc aaggcaacat gcagagggag ccaaaccagg
     1861 ccttcggttc tggaaataac tcttatagtg gctctaattc tggtgcagca attggttggg
     1921 gatcagcatc caatgcaggg tcgggcagtg gttttaatgg aggctttggc tcaagcatgg
     1981 attctaagtc ttctggctgg ggaatgtaga agcttgcggc cgcataatgc ttaagtcgaa
     2041 cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg
     2101 gaattgtgag cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat
     2161 atacatatgg cagatctcaa ttggatatcg gccggccacg cgatcgctga cgtcggtacc
     2221 ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct
     2281 actagcgcag cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc
     2341 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     2401 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2461 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2521 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2581 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2641 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2701 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2761 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2821 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttct ggcggcacga
     2881 tggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     2941 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3001 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3061 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3121 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3181 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3241 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3301 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3361 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3421 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3481 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3541 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3601 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3661 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     3721 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     3781 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     3841 catactcttc ctttttcaat catgattgaa gcatttatca gggttattgt ctcatgagcg
     3901 gatacatatt tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa aatcccttaa
     3961 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4021 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4081 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4141 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4201 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4261 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4321 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4381 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4441 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4501 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4561 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4621 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4681 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4741 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4801 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     4861 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4921 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4981 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5041 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     5101 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     5161 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     5221 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     5281 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     5341 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     5401 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     5461 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     5521 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     5581 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     5641 ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca
     5701 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5761 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5821 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5881 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     5941 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     6001 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     6061 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     6121 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     6181 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     6241 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     6301 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     6361 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     6421 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     6481 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     6541 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6601 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6661 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6721 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6781 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6841 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     6901 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     6961 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     7021 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     7081 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     7141 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     7201 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc
     7261 gcgaaattaa tacgactcac tatagg
//
