LOCUS       sequence-466578-        6364 bp    DNA     circular SYN 15-APR-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 6364)
  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 6364)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 R104A
FEATURES             Location/Qualifiers
     source          1..6364
                     /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(2414..2436)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2581..2599)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2667..2771
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2772..2840,2841..3632)
                     /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(2990..3009)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3803..4391
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4292..4311
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4545..4562
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4635..4712
                     /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             4713..5795
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4732..4751)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5808..5829
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5844..5849,5850..5867,5868..5874)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5882..5898
                     /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     5906..5922
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5906..5922
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5918..6364,1..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(5938..5955)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5938..5954)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5947..5969)
                     /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 tctctgacag
     1261 ctgttgacct gtcttcctct gttctgccgg gtgactccgt tggtctggcc gtaattctgc
     1321 acaccgactc tcgtaaagac tccccgccgg ctggttctcc ggcgggtcgc tctatctaca
     1381 actctttcta cgtgtactgc aaaggtccgt gccagcgtgt acagccgggt aaactgcgtg
     1441 ttcagtgttc cacttgccgt caggcgaccc tgaccctgac ccagggtccg agctgctggg
     1501 acgacgttct gatcccgaac cgtatgtccg gtgaatgtca gtccccgcac tgtccgggta
     1561 cctccgcaga atttttcttt aaatgcggtg cccacccgac ctctgacaaa gaaacctctg
     1621 ttgctctgca cctgatcgct accaactccc gtaacatcac ctgcatcacc tgcaccgacg
     1681 ttcgctcccc ggttctggtt tttcagtgca actctcgtca cgttatctgc ctggattgct
     1741 tccacctgta ctgcgttact cgtctgaatg accgccagtt cgttcacgac ccgcagctgg
     1801 gctactctct gccgtgcgtt gcgggttgcc cgaactccct gattaaagaa ctgcaccact
     1861 tccgtatcct gggcgaggaa cagtacaacc gttaccagca gtacggcgct gaagaatgcg
     1921 ttctgcagat gggtggcgtt ctgtgcccgc gtccgggttg cggcgcgggc ctgctgccgg
     1981 aaccggacca gcgtaaagtg acgtgtgaag gtggtaacgg tttaggttgc ggcttcgcgt
     2041 tctgtcgtga atgcaaagaa gcatatcatg aaggtgaatg ttccgcagtt tttgaagcta
     2101 gcggcaccac tacccaggcg taccgtgttg atgaacgtgc tgctgaacag gctcgttggg
     2161 aagcagcaag taaagaaact attaaaaaga ccactaaacc gtgtccgcgt tgccacgttc
     2221 cggttgaaaa gaacggtggt tgcatgcaca tgaaatgccc gcagccgcag tgccgtctgg
     2281 aatggtgctg gaactgcggt tgcgaatgga accgtgtttg catgggtgac cactggttcg
     2341 acgtttagct cgagcggccg catcgtgact gactgacgat ctgcctcgcg cgtttcggtg
     2401 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     2461 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     2521 gcgcagccat gacccagtca cgtagcgata gcggagtgta taattcttga agacgaaagg
     2581 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     2641 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2701 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2761 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2821 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2881 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2941 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3001 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3061 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3121 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3181 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3241 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3301 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3361 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3421 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3481 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3541 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3601 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3661 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3721 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3781 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3841 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3901 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3961 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4021 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4081 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4141 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4201 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4261 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4321 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4381 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4441 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4501 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4561 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4621 accgcataaa ttccgacacc atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc
     4681 gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg
     4741 cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg
     4801 tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca
     4861 accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca
     4921 gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac
     4981 tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg
     5041 cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg
     5101 accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg
     5161 tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg
     5221 gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa
     5281 gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa
     5341 ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca
     5401 tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg
     5461 cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg
     5521 tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtca accaccatca
     5581 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg
     5641 gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc
     5701 tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     5761 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     5821 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     5881 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cggattcact
     5941 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     6001 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     6061 ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt gcctggtttc cggcaccaga
     6121 agcggtgccg gaaagctggc tggagtgcga tcttcctgag gccgatactg tcgtcgtccc
     6181 ctcaaactgg cagatgcacg gttacgatgc gcccatctac accaacgtaa cctatcccat
     6241 tacggtcaat ccgccgtttg ttcccacgga gaatccgacg ggttgttact cgctcacatt
     6301 taatgttgat gaaagctggc tacaggaagg ccagacgcga attatttttg atggcgttgg
     6361 aatt
//
