LOCUS       sequence-171321-        5964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression, GST-fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5964)
  AUTHORS   Toyota R, Honjo Y, Imajo R, Satoh A
  TITLE     S-nitrosylation of laforin inhibits its phosphatase activity and is 
            implicated in Lafora disease
  JOURNAL   Sciencematters
REFERENCE   2  (bases 1 to 5964)
  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: 
            "Sciencematters"
COMMENT     Sequence Label: pGEX6P1-GST-Laforin
FEATURES             Location/Qualifiers
     source          1..5964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..654
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     612..634
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             661..684
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1757..1779)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1924..1942)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2010..2114
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2115..2183,2184..2975)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2333..2352)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3146..3734
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3635..3654
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3888..3905
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3978..4055
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4056..5138
                     /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(4075..4094)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5187..5192,5193..5210,5211..5217)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5225..5241
                     /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)."
     primer_bind     5249..5265
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5249..5265
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5261..5788
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5281..5298)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5281..5297)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5290..5312)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(5890..5895,5896..5911,5912..5918)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5926..5942
                     /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)."
ORIGIN
        1 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
       61 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
      121 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
      181 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
      241 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
      301 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
      361 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
      421 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
      481 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
      541 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
      601 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
      661 ctggaagttc tgttccaggg gcccctggga tccccgatgc gcttccgctt tggggtggtg
      721 gtgccacccg ccgtggccgg cgcccggccg gagctgctgg tggtggggtc gcggcccgag
      781 ctggggcgtt gggagccgcg cggtgccgtc cgcctgaggc cggccggcac cgcggcgggc
      841 gacggggccc tggcgctgca ggagccgggc ctgtggctcg gggaggtgga gctggcggcc
      901 gaggaggcgg cgcaggacgg ggcggagccg ggccgcgtgg acacgttctg gtacaagttc
      961 ctgaagcggg agccgggagg agagctctcc tgggaaggca atggacctca tcatgaccgt
     1021 tgctgtactt acaatgaaaa caacttggtg gatggtgtgt attgtctccc aataggacac
     1081 tggattgagg ccactggaca caccaatgaa atgaagcaca caacagactt ctattttaat
     1141 attgcaggcc accaagccat gcattattca agaattctac caaatatctg gctgggtagc
     1201 tgccctcgtc aggtggaaca tgtaaccatc aaactgaagc atgaattggg gattacagct
     1261 gtaatgaatt tccagactga atgggatatt gtacagaatt cctcaggctg taaccgctac
     1321 ccagagccca tgactccaga cactatgatt aaactatata gggaagaagg cttggcctac
     1381 atctggatgc caacaccaga tatgagcacc gaaggccgag tacagatgct gccccaggcg
     1441 gtgtgcctgc tgcatgcgct gctggagaag ggacacatcg tgtacgtgca ctgcaacgct
     1501 ggggtgggcc gctccaccgc ggctgtctgc ggctggctcc agtatgtgat gggctggaat
     1561 ctgaggaagg tgcagtattt cctcatggcc aagaggccgg ctgtctacat tgacgaagag
     1621 gccttggccc gggcacaaga agattttttc cagaaatttg ggaaggttcg ttcttctgtg
     1681 tgtagcctgt actcgagcgg ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg
     1741 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     1801 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     1861 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtataattct tgaagacgaa
     1921 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     1981 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2041 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2101 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2161 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2221 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2281 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2341 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2401 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2461 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2521 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2581 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2641 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     2701 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2761 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2821 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2881 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2941 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3001 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3061 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3121 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3181 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3241 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3301 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3361 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3421 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3481 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3541 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3601 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3661 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3721 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3781 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3841 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3901 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3961 cacaccgcat aaattccgac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     4021 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     4081 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     4141 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     4201 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     4261 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     4321 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     4381 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     4441 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     4501 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     4561 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     4621 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     4681 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     4741 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     4801 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     4861 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     4921 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     4981 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     5041 cgctagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5101 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     5161 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     5221 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacggattc
     5281 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5341 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5401 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc
     5461 agaagcggtg ccggaaagct ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt
     5521 cccctcaaac tggcagatgc acggttacga tgcgcccatc tacaccaacg tgacctatcc
     5581 cattacggtc aatccgccgt ttgttcccac ggagaatccg acgggttgtt actcgctcac
     5641 atttaatgtt gatgaaagct ggctacagga aggccagacg cgaattattt ttgatggcgt
     5701 tggaattagc ttatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga
     5761 agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca
     5821 ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga
     5881 aatgagctgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac
     5941 aatttcacac aggaaacagt attc
//
