LOCUS       sequence-212933-        7361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human Parkin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7361)
  AUTHORS   Wauer T, Komander D
  TITLE     Structure of the human Parkin ligase domain in an autoinhibited 
            state.
  JOURNAL   EMBO J. 2013 Jul 31;32(15):2099-112. doi: 10.1038/emboj.2013.125. 
            Epub 2013 May 31.
  PUBMED    23727886
REFERENCE   2  (bases 1 to 7361)
  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"; doi: 
            "10.1038/emboj.2013.125"; journalName: "EMBO J"; date: "2013-07-31- 
            31"; volume: "32"; issue: "15"; pages: "2099-112"
COMMENT     Sequence Label: Human Parkin (1-465)
FEATURES             Location/Qualifiers
     source          1..7361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..80
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    80..104
                     /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)."
     RBS             119..141
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             154..171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             172..825
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             844..867
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2286..2303
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2356..2374)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2370..2417
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2454..2909
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2541..2560)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2751..2772
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3001..3816)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3001..3816)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3724..3743
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3808..3827
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3938..4526
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4427..4446
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4680..4697
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4712..4851
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4777..4796)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4937..4959
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4953..5144)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5953..7035)
                     /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     6997..7016
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7036..7113)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7319..7338
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
       61 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      121 tgtttaactt taagaaggag atataccatg gcacatcacc atcaccatca catgtcccct
      181 atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat
      241 cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa atggcgaaac
      301 aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga tggtgatgtt
      361 aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa catgttgggt
      421 ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt ggatattaga
      481 tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt tgattttctt
      541 agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa aacatattta
      601 aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga tgttgtttta
      661 tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt
      721 gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc atggcctttg
      781 cagggctggc aagccacgtt tggtggtggc gaccatcctc caaaatcgga tctgagcagc
      841 ggtctggaag ttctgtttca gggcccgatg attgtttttg tgcgttttaa ctcgtcccac
      901 ggttttccgg ttgaagtcga tagcgatacc agcattttcc aactgaaaga agtcgtcgca
      961 aaacgtcagg gcgtgccggc agatcaactg cgtgtgattt ttgccggtaa agaactgcgc
     1021 aacgactgga ccgttcagaa ttgcgatctg gaccagcaaa gcattgttca tatcgtccag
     1081 cgtccgtggc gcaaaggcca agaaatgaac gcaaccggcg gtgatgaccc gcgtaatgca
     1141 gccggcggtt gtgaacgtga accgcagtct ctgacgcgcg ttgatctgag ctctagtgtc
     1201 ctgccgggtg acagtgtggg tctggcagtt attctgcaca ccgattcccg taaagactca
     1261 ccgccggcag gtagcccggc tggtcgttcg atctacaaca gcttctatgt gtactgcaaa
     1321 ggcccgtgtc agcgtgtcca accgggtaaa ctgcgcgtgc agtgctcgac ctgtcgtcaa
     1381 gcaacgctga ccctgacgca gggtccgagc tgctgggatg acgttctgat cccgaatcgt
     1441 atgtctggcg aatgccagag tccgcattgt ccgggtacga gcgcagaatt tttctttaaa
     1501 tgtggtgctc atccgacctc cgataaagaa acgtcagtgg cactgcacct gattgctacc
     1561 aacagtcgta atattacgtg catcacctgt acggatgttc gctctccggt gctggttttc
     1621 cagtgcaaca gtcgtcatgt catctgcctg gactgttttc acctgtactg tgttacccgt
     1681 ctgaatgatc gccagttcgt ccacgacccg caactgggct attccctgcc gtgcgttgcg
     1741 ggttgtccga actcactgat taaagaactg catcactttc gtatcctggg cgaagaacag
     1801 tataatcgct accagcaata tggtgcagaa gaatgcgtgc tgcagatggg cggtgttctg
     1861 tgcccgcgtc cgggttgtgg tgcaggtctg ctgccggaac cggatcagcg taaagtgacc
     1921 tgcgaaggcg gtaacggtct gggctgtggt ttcgcgtttt gccgcgaatg taaagaagcc
     1981 taccatgaag gcgaatgctc ggcggtcttc gaagccagcg gcaccacgac ccaggcatat
     2041 cgtgtggatg aacgcgcagc tgaacaagct cgctgggaag cggcctctaa agaaaccatt
     2101 aagaaaacca ccaaaccgtg cccgcgttgt catgtcccgg tggagaaaaa cggcggttgc
     2161 atgcacatga aatgtccgca gccgcaatgc cgtctggaat ggtgttggaa ctgcggctgc
     2221 gaatggaatc gtgtgtgtat gggtgaccac tggtttgatg tgtaaagctt tctagaccat
     2281 ttaaacacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     2341 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2401 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     2461 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2521 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2581 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2641 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2701 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2761 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2821 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2881 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     2941 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     3001 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     3061 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     3121 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     3181 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     3241 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     3301 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     3361 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     3421 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     3481 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     3541 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     3601 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     3661 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     3721 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     3781 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     3841 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     3901 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3961 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4021 aagagctaac aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4081 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4141 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4201 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4261 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4321 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4381 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4441 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4501 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4561 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4621 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4681 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4741 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4801 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4861 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4921 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4981 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     5041 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     5101 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     5161 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     5221 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     5281 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     5341 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5401 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     5461 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5521 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5581 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     5641 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     5701 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     5761 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     5821 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5881 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     5941 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6001 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     6061 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     6121 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     6181 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     6241 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     6301 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6361 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6421 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6481 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6541 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6601 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6661 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6721 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6781 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6841 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6901 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     6961 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     7021 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     7081 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     7141 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     7201 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     7261 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     7321 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc g
//
