LOCUS       sequence-64623-e        6136 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 6136)
  AUTHORS   Trempe JF, Sauve V, Grenier K, Seirafi M, Tang MY, Menade M, 
            Al-Abdul-Wahid S, Krett J, Wong K, Kozlov G, Nagar B, Fon EA, 
            Gehring K
  TITLE     Structure of Parkin Reveals Mechanisms for Ubiquitin Ligase 
            Activation.
  JOURNAL   Science. 2013 May 9.
  PUBMED    23661642
REFERENCE   2  (bases 1 to 6136)
  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: "Science. 
            2013 May 9."
COMMENT     Sequence Label: pGEX-parkin 77-465
FEATURES             Location/Qualifiers
     source          1..6136
                     /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(2186..2208)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2353..2371)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2439..2543
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2544..2612,2613..3404)
                     /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(2762..2781)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3575..4163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4064..4083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4317..4334
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4407..4484
                     /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             4485..5567
                     /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(4504..4523)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5580..5601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5616..5621,5622..5639,5640..5646)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5654..5670
                     /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     5678..5694
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5678..5694
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5690..6136,1..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(5710..5727)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5710..5726)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5719..5741)
                     /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 tctcacgaaa
      961 ccaacgcgtc tggtggtgac aaaccgcagt ctaccccgga aggttctatc tgggaaccgc
     1021 gttctctgac ccgtgttgac ctgtcttctc acatcctgcc ggcggactct gttggtctgg
     1081 cggttatcct ggacaccgac tctaaatctg actctgaagc ggcgcgtggt ccggcggcga
     1141 aaccgaccta ccactctttc ttcgtttact gcaaaggtcc gtgccacaaa gttcagccgg
     1201 gtaaactgcg tgttcagtgc ggtacctgcc gtcaggcgac cctgaccctg gcgcagggtc
     1261 cgtcttgctg ggacgacgtt ctgatcccga accgtatgtc tggtgaatgc cagtctccgg
     1321 actgcccggg tacccgtgcg gaattcttct tcaaatgcgg tgcgcacccg acctctgaca
     1381 aagacacctc tgttgcgctg aacctgatca ccaacaactc tcgttctatc ccgtgcatcg
     1441 cgtgcaccga cgttcgtaac ccggttctgg ttttccagtg caaccaccgt cacgttatct
     1501 gcctggactg cttccacctg tactgcgtta cccgtctgaa cgaccgtcag ttcgttcacg
     1561 acgcgcagct gggttactct ctgccgtgcg ttgcgggttg cccgaactct ctgatcaaag
     1621 aactgcacca cttccgtatc ctgggtgaag aacagtacaa ccgttaccag cagtacggtg
     1681 cggaagaatg cgttctgcag atgggtggtg ttctgtgccc gcgtccgggt tgcggtgcgg
     1741 gtctgctgcc ggaacagggt cagcgtaaag ttacctgcga aggtggtaac ggtctgggtt
     1801 gcggtttcgt tttctgccgt gactgcaaag aagcgtacca cgaaggtgaa tgcgactcta
     1861 tgttcgaagc gtctggtgcg acctctcagg cgtaccgtgt tgaccagcgt gcggcggaac
     1921 aggcgcgttg ggaagaagcg tctaaagaaa ccatcaaaaa aaccaccaaa ccgtgcccgc
     1981 gttgcaacgt tccgatcgaa aaaaacggtg gttgcatgca catgaaatgc ccgcagccgc
     2041 agtgcaaact ggaatggtgc tggaactgcg gttgcgaatg gaaccgtgcg tgcatgggtg
     2101 accactggtt cgacgtttag ctcgagcggc cgcatcgtga ctgactgacg atctgcctcg
     2161 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2221 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2281 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt
     2341 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2401 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2461 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2521 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2581 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2641 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2701 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2761 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     2821 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2881 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2941 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3001 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3061 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     3121 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3181 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3241 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3301 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3361 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3421 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3481 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3541 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3601 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3661 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3721 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3781 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3841 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3901 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3961 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4021 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4081 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4141 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4201 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4261 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4321 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4381 gcggtatttc acaccgcata aattccgaca ccatcgaatg gtgcaaaacc tttcgcggta
     4441 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4501 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4561 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4621 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4681 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4741 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     4801 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     4861 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     4921 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     4981 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5041 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5101 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5161 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5221 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5281 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     5341 caaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     5401 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     5461 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5521 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5581 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     5641 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     5701 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5761 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5821 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt
     5881 tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac
     5941 tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt
     6001 aacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta
     6061 ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt
     6121 tgatggcgtt ggaatt
//
