LOCUS       sequence-391156-        6321 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 6321)
  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 6321)
  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: pCold TDP43 WT
FEATURES             Location/Qualifiers
     source          1..6321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..81
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    84..100
                     /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)."
     5'UTR           122..255
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             256..270
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     CDS             271..288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             289..300
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             313..966
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     924..946
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             982..1002
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     3'UTR           2282..2426
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     primer_bind     complement(2467..2489)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2589..2608
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2625..3080)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2762..2783)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2974..2993
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3180..3284
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3285..3353,3354..4145)
                     /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     complement(3503..3522)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4316..4904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4805..4824
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5076..6158)
                     /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     6120..6139
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6159..6236)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa
       61 tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat
      121 ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt
      181 cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt
      241 aagaggtaat acaccatgaa tcacaaagtg catcatcatc atcatcatat cgaaggtagg
      301 catatggagc tcatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc
      361 actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat
      421 gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct
      481 tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct
      541 gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa
      601 ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa
      661 actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt
      721 ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat
      781 gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta
      841 gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc
      901 aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct
      961 ccaaaatcgg atctggctag cgagaatctt tattttcagg gatccatgtc tgaatatatt
     1021 cgggtaaccg aagatgagaa cgatgagccc attgaaatac catcggaaga cgatgggacg
     1081 gtgctgctct ccacggttac agcccagttt ccaggggcgt gtgggcttcg ctacaggaat
     1141 ccagtgtctc agtgtatgag aggtgtccgg ctggtagaag gaattctgca tgccccagat
     1201 gctggctggg gaaatctggt gtatgttgtc aactatccaa aagataacaa aagaaaaatg
     1261 gatgagacag atgcttcatc agcagtgaaa gtgaaaagag cagtccagaa aacatccgat
     1321 ttaatagtgt tgggtctccc atggaaaaca accgaacagg acctgaaaga gtattttagt
     1381 acctttggag aagttcttat ggtgcaggtc aagaaagatc ttaagactgg tcattcaaag
     1441 gggtttggct ttgttcgttt tacggaatat gaaacacaag tgaaagtaat gtcacagcga
     1501 catatgatag atggacgatg gtgtgactgc aaacttccta attctaagca aagccaagat
     1561 gagcctttga gaagcagaaa agtgtttgtg gggcgctgta cagaggacat gactgaggat
     1621 gagctgcggg agttcttctc tcagtacggg gatgtgatgg atgtcttcat ccccaagcca
     1681 ttcagggcct ttgcctttgt tacatttgca gatgatcaga ttgcgcagtc tctttgtgga
     1741 gaggacttga tcattaaagg aatcagcgtt catatatcca atgccgaacc taagcacaat
     1801 agcaatagac agttagaaag aagtggaaga tttggtggta atccaggtgg ctttgggaat
     1861 cagggtggat ttggtaatag cagagggggt ggagctggtt tgggaaacaa tcaaggtagt
     1921 aatatgggtg gtgggatgaa ctttggtgcg ttcagcatta atccagccat gatggctgcc
     1981 gcccaggcag cactacagag cagttggggt atgatgggca tgttagccag ccagcagaac
     2041 cagtcaggcc catcgggtaa taaccaaaac caaggcaaca tgcagaggga gccaaaccag
     2101 gccttcggtt ctggaaataa ctcttatagt ggctctaatt ctggtgcagc aattggttgg
     2161 ggatcagcat ccaatgcagg gtcgggcagt ggttttaatg gaggctttgg ctcaagcatg
     2221 gattctaagt cttctggctg gggaatgtag aagcttgtcg acctgcagtc tagataggta
     2281 atctctgctt aaaagcacag aatctaagat ccctgccatt tggcggggat ttttttattt
     2341 gttttcagga aataaataat cgatcgcgta ataaaatcta ttattatttt tgtgaagaat
     2401 aaatttgggt gcaatgagaa tgcgcaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     2461 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     2521 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     2581 taactatgcg gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa
     2641 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     2701 cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagggt tgagtgttgt
     2761 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     2821 aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg
     2881 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     2941 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     3001 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     3061 tgcgccgcta cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac
     3121 agatgcgtaa ggagaaaata ccgcatcagg cgtcaggtgg cacttttcgg ggaaatgtgc
     3181 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3241 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3301 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3361 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3421 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3481 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3541 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3601 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3661 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3721 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3781 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3841 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3901 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3961 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4021 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4081 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4141 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4201 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4261 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4321 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4381 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4441 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4501 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4561 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4621 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4681 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4741 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4801 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4861 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4921 cctttttacg gttcctggcc ttttgctggc cttttgctca catagtcatg ccccgcgccc
     4981 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5041 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5101 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5161 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5221 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5281 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5341 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     5401 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5461 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5521 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5581 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5641 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5701 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5761 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5821 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5881 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5941 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     6001 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     6061 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     6121 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     6181 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     6241 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     6301 gccgttgagc accgccgccg c
//
