LOCUS       sequence-212914-        7349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of PhPINK1 (biochemistry).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7349)
  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 7349)
  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 (115 - 575)
FEATURES             Location/Qualifiers
     source          1..7349
                     /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             302..319
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             320..973
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             992..1015
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2422..2439
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2492..2510)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2506..2553
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2590..3045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2677..2696)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2887..2908
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3137..3952)
                     /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(3137..3952)
                     /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     3860..3879
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3944..3963
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4074..4662
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4563..4582
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4816..4833
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4848..4987
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4913..4932)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5073..5095
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5089..5280)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6089..7171)
                     /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     7133..7152
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7172..7249)
                     /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 ataccatggc
      301 acatcaccat caccatcaca tgtcccctat actaggttat tggaaaatta agggccttgt
      361 gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga
      421 gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa
      481 tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta
      541 tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat
      601 gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga
      661 ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga
      721 agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat
      781 gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc
      841 aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa
      901 atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga
      961 ccatcctcca aaatcggatc tgagcagcgg tctggaagtt ctgtttcagg gcccgagcgg
     1021 tctgctgacc aaagatgatg aactggaagg tatttgttgg gaaattcgtg aagcagtgag
     1081 caaaggtaaa tggaatgata gcgaaagcga aaatgttgaa cagctgcaag cagcaaatct
     1141 ggatgagctg gatctgggtg aaccgattgc aaaaggttgt aatgcagttg tttatagcgc
     1201 caaactgaaa aatgtgcaga gcaataaact ggcacatcag ctggcagtta aaatgatgtt
     1261 taactatgac gtggaaagca acagcaccgc aattctgaaa gcaatgtatc gtgaaaccgt
     1321 tccggcaatg agctattttt tcaatcagaa cctgtttaac atcgagaaca tcagcgattt
     1381 caaaattcgt ctgcctccgc atccgaatat tgttcgtatg tatagcgttt ttgcagatcg
     1441 tattccggat ctgcagtgta ataaacagct gtatccggaa gcactgccac cgcgtattaa
     1501 tccggaaggt agtggtcgta atatgagcct gtttctggtt atgaaacgtt atgattgcac
     1561 cctgaaagaa tatctgcgtg ataaaacccc gaatatgcgt agcagcatcc tgctgctgag
     1621 ccagctgctg gaagcagttg cacatatgaa cattcataac atcagccacc gtgatctgaa
     1681 atccgataat attctggttg atctgagcga aggtgatgca tatccgacca ttgttattac
     1741 cgattttggt tgttgtctgt gcgataaaca gaatggcctg gttattccgt atcgtagcga
     1801 agatcaggat aaaggtggta atcgtgcact gatggcaccg gaaattgcaa atgcaaaacc
     1861 gggtacattt agctggctga actacaaaaa aagcgatctg tgggcagttg gtgcaattgc
     1921 ctatgaaatc ttcaacatcg ataacccgtt ctatgacaaa accatgaaac tgctgtccaa
     1981 aagctataaa gaagaagatc tgccggaact gccggatacc attccgttta ttatccgtaa
     2041 tctggtgagc aatatgctga gccgtagcac caataaacgt ctggattgtg atgttgcagc
     2101 aaccgttgca cagctgtacc tgtgggcacc gagcagttgg ctgaaagaaa actataccct
     2161 gccgaatagc aacgaaatta ttcagtggct gctgtgtctg agcagcaaag ttctgtgtga
     2221 acgtgatatt accgcacgca ataaaaccaa taccatgagc gaaagtgtta gcaaagcaca
     2281 gtataaaggt cgtcgtagcc tgccggaata tgaactgatt gccagctttc tgcgtcgtgt
     2341 tcgtctgcat ctggttcgta aaggcctgaa atggattcaa gaactgcaca tctataacta
     2401 aagctttcta gaccatttaa acaccaccac caccaccact gagatccggc tgctaacaaa
     2461 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2521 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2581 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2641 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2701 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2761 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2821 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2881 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2941 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3001 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     3061 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3121 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     3181 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     3241 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     3301 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     3361 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     3421 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     3481 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     3541 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     3601 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     3661 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3721 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3781 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3841 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3901 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3961 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     4021 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4081 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4141 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4201 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     4261 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4321 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4381 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4441 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4501 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4561 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4621 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4681 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4741 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4801 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4861 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4921 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4981 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5041 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5101 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     5161 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     5221 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     5281 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     5341 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     5401 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     5461 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5521 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5581 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5641 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5701 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5761 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5821 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5881 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5941 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     6001 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     6061 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6121 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6181 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     6241 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     6301 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     6361 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6421 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6481 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6541 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6601 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6661 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6721 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6781 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6841 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6901 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6961 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     7021 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     7081 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     7141 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     7201 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     7261 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     7321 agcaccgccg ccgcaaggaa tggtgcatg
//
