LOCUS       sequence-219778-        6869 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of PhPINK1 (crystallography).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6869)
  AUTHORS   Schubert AF, Gladkova C, Pardon E, Wagstaff JL, Freund SMV, Steyaert
            J, Maslen SL, Komander D
  TITLE     Structure of PINK1 in complex with its substrate ubiquitin.
  JOURNAL   Nature. 2017 Dec 7;552(7683):51-56. doi: 10.1038/nature24645. Epub 
            2017 Oct 30.
  PUBMED    29160309
REFERENCE   2  (bases 1 to 6869)
  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/nature24645"; journalName: "Nature"; date: "2017-12-7- 7"; 
            volume: "552"; issue: "7683"; pages: "51-56"
COMMENT     Sequence Label: Pediculus humanus corporis PINK1 (143 - 575)
FEATURES             Location/Qualifiers
     source          1..6869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1942..1959
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2012..2030)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2026..2073
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2110..2565
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2197..2216)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2407..2428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2657..3472)
                     /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(2657..3472)
                     /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     3380..3399
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3464..3483
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3594..4182
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4083..4102
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4336..4353
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4368..4507
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4433..4452)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4593..4615
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4609..4800)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5609..6691)
                     /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     6653..6672
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6692..6769)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 tagcagccat caccatcatc atcacgggag cgatagcgaa gtgaaccagg aagcgaaacc
      361 ggaagttaaa ccggaagtga aaccggaaac ccatattaat ctgaaagtta gcgacggcag
      421 cagcgaaatc ttttttaaaa ttaaaaaaac caccccgctg cgtcgcctga tggaagcctt
      481 tgcgaaacgt cagggtaaag aaatggatag cctgcgcttt ctgtatgacg gcatccgtat
      541 tcaggccgat cagaccccgg aagacctgga tatggaagac aacgatatta ttgaagcgca
      601 tcgcgaacag atcggtggta gcgaaagcga aaatgttgaa cagctgcaag cagcaaatct
      661 ggatgagctg gatctgggtg aaccgattgc aaaaggttgt aatgcagttg tttatagcgc
      721 caaactgaaa aatgtgcaga gcaataaact ggcacatcag ctggcagtta aaatgatgtt
      781 taactatgac gtggaaagca acagcaccgc aattctgaaa gcaatgtatc gtgaaaccgt
      841 tccggcaatg agctattttt tcaatcagaa cctgtttaac atcgagaaca tcagcgattt
      901 caaaattcgt ctgcctccgc atccgaatat tgttcgtatg tatagcgttt ttgcagatcg
      961 tattccggat ctgcagtgta ataaacagct gtatccggaa gcactgccac cgcgtattaa
     1021 tccggaaggt agtggtcgta atatgagcct gtttctggtt atgaaacgtt atgattgcac
     1081 cctgaaagaa tatctgcgtg ataaaacccc gaatatgcgt agcagcatcc tgctgctgag
     1141 ccagctgctg gaagcagttg cacatatgaa cattcataac atcagccacc gtgatctgaa
     1201 atccgataat attctggttg atctgagcga aggtgatgca tatccgacca ttgttattac
     1261 cgattttggt tgttgtctgt gcgataaaca gaatggcctg gttattccgt atcgtagcga
     1321 agatcaggat aaaggtggta atcgtgcact gatggcaccg gaaattgcaa atgcaaaacc
     1381 gggtacattt agctggctga actacaaaaa aagcgatctg tgggcagttg gtgcaattgc
     1441 ctatgaaatc ttcaacatcg ataacccgtt ctatgacaaa accatgaaac tgctgtccaa
     1501 aagctataaa gaagaagatc tgccggaact gccggatacc attccgttta ttatccgtaa
     1561 tctggtgagc aatatgctga gccgtagcac caataaacgt ctggattgtg atgttgcagc
     1621 aaccgttgca cagctgtacc tgtgggcacc gagcagttgg ctgaaagaaa actataccct
     1681 gccgaatagc aacgaaatta ttcagtggct gctgtgtctg agcagcaaag ttctgtgtga
     1741 acgtgatatt accgcacgca ataaaaccaa taccatgagc gaaagtgtta gcaaagcaca
     1801 gtataaaggt cgtcgtagcc tgccggaata tgaactgatt gccagctttc tgcgtcgtgt
     1861 tcgtctgcat ctggttcgta aaggcctgaa atggattcaa gaactgcaca tctataacta
     1921 aagctttcta gaccatttaa acaccaccac caccaccact gagatccggc tgctaacaaa
     1981 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2041 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2101 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2161 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2221 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2281 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2341 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2401 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2461 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2521 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     2581 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2641 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2701 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2761 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2821 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2881 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2941 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     3001 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     3061 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     3121 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     3181 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3241 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3301 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3361 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3421 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3481 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     3541 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3601 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3661 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3721 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3781 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3841 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3901 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3961 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4021 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4081 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4141 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4201 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4261 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4321 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4381 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4441 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4501 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4561 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4621 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4681 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4741 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4801 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4861 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4921 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4981 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5041 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5101 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5161 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5221 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5281 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5341 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5401 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5461 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5521 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5581 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5641 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5701 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5761 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     5821 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     5881 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     5941 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6001 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6061 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6121 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6181 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6241 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6301 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6361 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6421 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6481 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6541 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6601 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6661 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6721 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     6781 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     6841 agcaccgccg ccgcaaggaa tggtgcatg
//
