LOCUS       sequence-64626-e        5944 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 5944)
  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 5944)
  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 141-465
FEATURES             Location/Qualifiers
     source          1..5944
                     /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(1994..2016)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2161..2179)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2247..2351
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2352..2420,2421..3212)
                     /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(2570..2589)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3383..3971
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3872..3891
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4125..4142
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4215..4292
                     /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             4293..5375
                     /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(4312..4331)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5388..5409
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5424..5429,5430..5447,5448..5454)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5462..5478
                     /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     5486..5502
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5486..5502
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5498..5944,1..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(5518..5535)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5518..5534)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5527..5549)
                     /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 ccgacctacc
      961 actctttctt cgtttactgc aaaggtccgt gccacaaagt tcagccgggt aaactgcgtg
     1021 ttcagtgcgg tacctgccgt caggcgaccc tgaccctggc gcagggtccg tcttgctggg
     1081 acgacgttct gatcccgaac cgtatgtctg gtgaatgcca gtctccggac tgcccgggta
     1141 cccgtgcgga attcttcttc aaatgcggtg cgcacccgac ctctgacaaa gacacctctg
     1201 ttgcgctgaa cctgatcacc aacaactctc gttctatccc gtgcatcgcg tgcaccgacg
     1261 ttcgtaaccc ggttctggtt ttccagtgca accaccgtca cgttatctgc ctggactgct
     1321 tccacctgta ctgcgttacc cgtctgaacg accgtcagtt cgttcacgac gcgcagctgg
     1381 gttactctct gccgtgcgtt gcgggttgcc cgaactctct gatcaaagaa ctgcaccact
     1441 tccgtatcct gggtgaagaa cagtacaacc gttaccagca gtacggtgcg gaagaatgcg
     1501 ttctgcagat gggtggtgtt ctgtgcccgc gtccgggttg cggtgcgggt ctgctgccgg
     1561 aacagggtca gcgtaaagtt acctgcgaag gtggtaacgg tctgggttgc ggtttcgttt
     1621 tctgccgtga ctgcaaagaa gcgtaccacg aaggtgaatg cgactctatg ttcgaagcgt
     1681 ctggtgcgac ctctcaggcg taccgtgttg accagcgtgc ggcggaacag gcgcgttggg
     1741 aagaagcgtc taaagaaacc atcaaaaaaa ccaccaaacc gtgcccgcgt tgcaacgttc
     1801 cgatcgaaaa aaacggtggt tgcatgcaca tgaaatgccc gcagccgcag tgcaaactgg
     1861 aatggtgctg gaactgcggt tgcgaatgga accgtgcgtg catgggtgac cactggttcg
     1921 acgtttagct cgagcggccg catcgtgact gactgacgat ctgcctcgcg cgtttcggtg
     1981 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     2041 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     2101 gcgcagccat gacccagtca cgtagcgata gcggagtgta taattcttga agacgaaagg
     2161 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     2221 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2281 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2341 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2401 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2461 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2521 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2581 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2641 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2701 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2761 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2821 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2881 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2941 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3001 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3061 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3121 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3181 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3241 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3301 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3361 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3421 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3481 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3541 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3601 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3661 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3721 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3781 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3841 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3901 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3961 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4021 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4081 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4141 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4201 accgcataaa ttccgacacc atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc
     4261 gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg
     4321 cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg
     4381 tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca
     4441 accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca
     4501 gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac
     4561 tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg
     4621 cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg
     4681 accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg
     4741 tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg
     4801 gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa
     4861 gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa
     4921 ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca
     4981 tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg
     5041 cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg
     5101 tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtca accaccatca
     5161 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg
     5221 gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc
     5281 tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     5341 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     5401 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     5461 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cggattcact
     5521 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     5581 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     5641 ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt gcctggtttc cggcaccaga
     5701 agcggtgccg gaaagctggc tggagtgcga tcttcctgag gccgatactg tcgtcgtccc
     5761 ctcaaactgg cagatgcacg gttacgatgc gcccatctac accaacgtaa cctatcccat
     5821 tacggtcaat ccgccgtttg ttcccacgga gaatccgacg ggttgttact cgctcacatt
     5881 taatgttgat gaaagctggc tacaggaagg ccagacgcga attatttttg atggcgttgg
     5941 aatt
//
