LOCUS       sequence-450999-        6337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of parkin with a mutated ACT..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6337)
  AUTHORS   Huq TS, Luo J, Fakih R, Sauve V, Gehring K
  TITLE     Naturally occurring hyperactive variants of human parkin.
  JOURNAL   Commun Biol. 2024 Aug 8;7(1):961. doi: 10.1038/s42003-024-06656-x.
  PUBMED    39117722
REFERENCE   2  (bases 1 to 6337)
  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: "Commun 
            Biol."; date: "2024-08-8"; volume: "7(1):961. doi"; pages: " 
            10.1038/s42003-024-06656-x"
COMMENT     Sequence Label: pGEX-parkin delta 101-109
FEATURES             Location/Qualifiers
     source          1..6337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2387..2409)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2554..2572)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2640..2744
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2745..2813,2814..3605)
                     /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(2963..2982)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3776..4364
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4265..4284
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4518..4535
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4608..4685
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4686..5768
                     /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(4705..4724)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5781..5802
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5817..5822,5823..5840,5841..5847)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5855..5871
                     /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     5879..5895
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5879..5895
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5891..6337,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5911..5928)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5911..5927)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5920..5942)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc atgatcgttt
      961 tcgttcgttt caactcttct cacggtttcc cggttgaagt tgactctgac acctctatct
     1021 tccagctgaa agaagttgtt gcgaaacgtc agggtgttcc agcagaccag ctgcgtgtta
     1081 tcttcgcagg taaagaactg cgtaacgact ggaccgttca gaactgcgat ctggatcagc
     1141 agtctatcgt tcacatcgtt cagcgtccgt ggcgtaaagg tcaggaaatg aacgcgactg
     1201 gtggtgacga cccgcgtaac gcggcgggtg gttgtgaacg tgaaccgcag tctgttctgc
     1261 cgggtgactc cgttggtctg gccgtaattc tgcacaccga ctctcgtaaa gactccccgc
     1321 cggctggttc tccggcgggt cgctctatct acaactcttt ctacgtgtac tgcaaaggtc
     1381 cgtgccagcg tgtacagccg ggtaaactgc gtgttcagtg ttccacttgc cgtcaggcga
     1441 ccctgaccct gacccagggt ccgagctgct gggacgacgt tctgatcccg aaccgtatgt
     1501 ccggtgaatg tcagtccccg cactgtccgg gtacctccgc agaatttttc tttaaatgcg
     1561 gtgcccaccc gacctctgac aaagaaacct ctgttgctct gcacctgatc gctaccaact
     1621 cccgtaacat cacctgcatc acctgcaccg acgttcgctc cccggttctg gtttttcagt
     1681 gcaactctcg tcacgttatc tgcctggatt gcttccacct gtactgcgtt actcgtctga
     1741 atgaccgcca gttcgttcac gacccgcagc tgggctactc tctgccgtgc gttgcgggtt
     1801 gcccgaactc cctgattaaa gaactgcacc acttccgtat cctgggcgag gaacagtaca
     1861 accgttacca gcagtacggc gctgaagaat gcgttctgca gatgggtggc gttctgtgcc
     1921 cgcgtccggg ttgcggcgcg ggcctgctgc cggaaccgga ccagcgtaaa gtgacgtgtg
     1981 aaggtggtaa cggtttaggt tgcggcttcg cgttctgtcg tgaatgcaaa gaagcatatc
     2041 atgaaggtga atgttccgca gtttttgaag ctagcggcac cactacccag gcgtaccgtg
     2101 ttgatgaacg tgctgctgaa caggctcgtt gggaagcagc aagtaaagaa actattaaaa
     2161 agaccactaa accgtgtccg cgttgccacg ttccggttga aaagaacggt ggttgcatgc
     2221 acatgaaatg cccgcagccg cagtgccgtc tggaatggtg ctggaactgc ggttgcgaat
     2281 ggaaccgtgt ttgcatgggt gaccactggt tcgacgttta gctcgagcgg ccgcatcgtg
     2341 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     2401 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     2461 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     2521 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     2581 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2641 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2701 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2761 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2821 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2881 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2941 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     3001 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3061 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3121 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3181 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3241 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     3301 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3361 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3421 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3481 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3541 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3601 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3661 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3721 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3781 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3841 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3901 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3961 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4021 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4081 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4141 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4201 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4261 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4321 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4381 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4441 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4501 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4561 attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat
     4621 ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     4681 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     4741 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     4801 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     4861 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     4921 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     4981 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     5041 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     5101 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     5161 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     5221 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     5281 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     5341 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     5401 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     5461 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     5521 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     5581 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     5641 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     5701 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5761 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     5821 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     5881 ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg
     5941 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     6001 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     6061 cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg
     6121 cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga
     6181 tgcgcccatc tacaccaacg taacctatcc cattacggtc aatccgccgt ttgttcccac
     6241 ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga
     6301 aggccagacg cgaattattt ttgatggcgt tggaatt
//
