LOCUS       sequence-212910-        7109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human USP30 (construct 8).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7109)
  AUTHORS   Gersch M, Gladkova C, Schubert AF, Michel MA, Maslen S, Komander D
  TITLE     Mechanism and regulation of the Lys6-selective deubiquitinase USP30.
  JOURNAL   Nat Struct Mol Biol. 2017 Sep 25. doi: 10.1038/nsmb.3475.
  PUBMED    28945249
REFERENCE   2  (bases 1 to 7109)
  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: "Nat Struct 
            Mol Biol. 2017 Sep 25. doi: 10.1038/nsmb.3475."
COMMENT     Sequence Label: Human USP30 (64-502, construct 8, MG-26-21)
FEATURES             Location/Qualifiers
     source          1..7109
                     /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             335..358
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             359..1012
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             1031..1054
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2182..2199
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2266..2313
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2350..2805
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2437..2456)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2647..2668
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2897..3712)
                     /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(2897..3712)
                     /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     3620..3639
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3704..3723
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3834..4422
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4323..4342
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4576..4593
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4608..4747
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4673..4692)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4833..4855
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4849..5040)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5849..6931)
                     /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     6893..6912
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6932..7009)
                     /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 cagcagccat catcatcatc atcacagcag cggtctggaa gttctgtttc agggcccgat
      361 gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt
      421 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg
      481 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg
      541 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat
      601 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga
      661 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga
      721 ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac
      781 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt
      841 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa
      901 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg
      961 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct
     1021 gagcagcggt ctggaagttc tgtttcaggg cccgaaaggt ctggttccgg gtctggttaa
     1081 tctgggtaat acctgtttta tgaatagcct gctgcagggt ctgagcgcat gtccggcatt
     1141 tattcgttgg ctggaagaat tcaccagcca gtatagccgt gatcagaaag aaccgcctag
     1201 ccatcagtat ctgagcctga ccctgctgca tctgctgaaa gcactgagct gtcaagaggt
     1261 taccgatgat gaagttctgg atgcaagctg tctgctggat gttctgcgta tgtatcgttg
     1321 gcagattagc agctttgaag aacaggatgc acatgaactg tttcatgtta ttaccagcag
     1381 cctggaagat gaacgtgatc gtcagcctcg tgttacccac ctgtttgatg ttcatagcct
     1441 ggaacagcag agcgaaatta ccccgaaaca aattacctgt cgtacccgtg gtagtccgca
     1501 tccgaccagc aatcattgga aaagccagca tccgtttcat ggtcgtctga ccagtaatat
     1561 ggtttgtaaa cattgcgaac atcagagtcc ggttcgtttt gatacctttg atagcctgag
     1621 cctgtcaatt ccggcagcaa cctggggtca tccgctgacc ctggatcatt gtctgcatca
     1681 tttcattagc agcgaaagcg ttcgtgatgt tgtttgtgat aactgcacca aaattgaagc
     1741 aaaaggcacc ctgaatggtg aaaaagttga acatcagcgt accacctttg tgaaacagct
     1801 gaaactgggt aaactgccgc agtgtctgtg tattcatctg cagcgtctgt cttggagcag
     1861 ccatggtaca ccgctgaaac gtcatgaaca tgtgcagttt aatgaggacc tggatatgga
     1921 cgagtataaa taccactcca atgcgagtac ctacctgttt cgtctgatgg cagttgttgt
     1981 tcatcatggt gatatgcata gcggtcattt tgttacctat cgtcgtagcc ctccgagcgc
     2041 acgtaatccg ctgagcacca gtaatcagtg gctgtgggtt agtgatgata ccgttcgtaa
     2101 agcaagcctg caagaagttc tgagcagcag cgcatatctg ctgttttacg agcgtgttta
     2161 aagctttcta gaccatttaa acaccaccac caccaccact gagatccggc tgctaacaaa
     2221 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2281 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2341 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2401 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2461 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2521 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2581 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2641 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2701 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2761 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     2821 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2881 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2941 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     3001 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     3061 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     3121 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     3181 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     3241 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     3301 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     3361 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     3421 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3481 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3541 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3601 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3661 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3721 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     3781 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3841 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3901 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3961 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     4021 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4081 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4141 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4201 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4261 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4321 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4381 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4441 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4501 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4561 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4621 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4681 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4741 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4801 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4861 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4921 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4981 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     5041 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     5101 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     5161 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     5221 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5281 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5341 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5401 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5461 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5521 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5581 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5641 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5701 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5761 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5821 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5881 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5941 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     6001 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     6061 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     6121 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6181 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6241 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6301 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6361 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6421 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6481 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6541 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6601 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6661 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6721 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6781 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6841 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6901 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6961 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     7021 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     7081 agcaccgccg ccgcaaggaa tggtgcatg
//
