LOCUS       sequence-64627-e        5707 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 5707)
  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 5707)
  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 219-465
FEATURES             Location/Qualifiers
     source          1..5707
                     /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(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 (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             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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4075..4094)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5151..5172
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     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             join(5261..5707,1..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(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"
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 aaagacacct
      961 ctgttgcgct gaacctgatc accaacaact ctcgttctat cccgtgcatc gcgtgcaccg
     1021 acgttcgtaa cccggttctg gttttccagt gcaaccaccg tcacgttatc tgcctggact
     1081 gcttccacct gtactgcgtt acccgtctga acgaccgtca gttcgttcac gacgcgcagc
     1141 tgggttactc tctgccgtgc gttgcgggtt gcccgaactc tctgatcaaa gaactgcacc
     1201 acttccgtat cctgggtgaa gaacagtaca accgttacca gcagtacggt gcggaagaat
     1261 gcgttctgca gatgggtggt gttctgtgcc cgcgtccggg ttgcggtgcg ggtctgctgc
     1321 cggaacaggg tcagcgtaaa gttacctgcg aaggtggtaa cggtctgggt tgcggtttcg
     1381 ttttctgccg tgactgcaaa gaagcgtacc acgaaggtga atgcgactct atgttcgaag
     1441 cgtctggtgc gacctctcag gcgtaccgtg ttgaccagcg tgcggcggaa caggcgcgtt
     1501 gggaagaagc gtctaaagaa accatcaaaa aaaccaccaa accgtgcccg cgttgcaacg
     1561 ttccgatcga aaaaaacggt ggttgcatgc acatgaaatg cccgcagccg cagtgcaaac
     1621 tggaatggtg ctggaactgc ggttgcgaat ggaaccgtgc gtgcatgggt gaccactggt
     1681 tcgacgttta gctcgagcgg 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 ggtgcaaaac 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 tcaaccacca
     4921 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     4981 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     5041 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5101 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     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 taacctatcc
     5581 cattacggtc aatccgccgt ttgttcccac ggagaatccg acgggttgtt actcgctcac
     5641 atttaatgtt gatgaaagct ggctacagga aggccagacg cgaattattt ttgatggcgt
     5701 tggaatt
//
