LOCUS       sequence-218962-        7064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human USP30 (construct 13i).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7064)
  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 7064)
  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 13i, C77A, MG-3...
FEATURES             Location/Qualifiers
     source          1..7064
                     /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             365..382
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             392..415
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             416..1069
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             1088..1111
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2137..2154
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2221..2268
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2305..2760
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2392..2411)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2602..2623
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2852..3667)
                     /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(2852..3667)
                     /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     3575..3594
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3659..3678
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3789..4377
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4278..4297
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4531..4548
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4563..4702
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4628..4647)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4788..4810
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4804..4995)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5804..6886)
                     /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     6848..6867
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6887..6964)
                     /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 ggcacatcac catcaccatc acagcagcgg tctggaagtt ctgtttcagg gcccgatgtc
      421 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
      481 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
      541 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
      601 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
      661 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
      721 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
      781 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
      841 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
      901 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
      961 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
     1021 tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat cggatctgag
     1081 cagcggtctg gaagttctgt ttcagggccc gaaaggtctg gttccgggtc tggttaatct
     1141 gggtaatacc gcttttatga atagcctgct gcagggtctg agcgcatgtc cggcatttat
     1201 tcgttggctg gaagaattca ccagccagta tagccgtgat cagaaagaac cgcctagcca
     1261 tcagtatctg agcctgaccc tgctgcatct gctgaaagca ctgagctgtc aagaggttac
     1321 cgatgatgaa gttctggatg caagctgtct gctggatgtt ctgcgtatgt atcgttggca
     1381 gattagcagc tttgaagaac aggatgcaca tgaactgttt catgttatta ccagcagcct
     1441 ggaagatgaa cgtgatggtt ccggctctca ttggaaaagc cagcatccgt ttcatggtcg
     1501 tctgaccagt aatatggttt gtaaacattg cgaacatcag agtccggttc gttttgatac
     1561 ctttgatagc ctgagcctgt caattccggc agcaacctgg ggtcatccgc tgaccctgga
     1621 tcattgtctg catcatttca ttagcagcga aagcgttcgt gatgttgttt gtgataactg
     1681 caccaaaatt gaagcaaaag gcaccctgaa tggtgaaaaa gttgaacatc agcgtaccac
     1741 ctttgtgaaa cagctgaaac tgggtaaact gccgcagtgt ctgtgtattc atctgcagcg
     1801 tctgtcttgg agcagccatg gtacaccgct gaaacgtcat gaacatgtgc agtttaatga
     1861 ggacctgagc atggacgagt ataaatacca ctccaatgcg agtacctacc tgtttcgtct
     1921 gatggcagtt gttgttcatc atggtgatat gcatagcggt cattttgtta cctatcgtcg
     1981 tagccctccg agcgcacgta atccgctgag caccagtaat cagtggctgt gggttagtga
     2041 tgataccgtt cgtaaagcaa gcctgcaaga agttctgagc agcagcgcat atctgctgtt
     2101 ttacgagcgt gtttaaagct ttctagacca tttaaacacc accaccacca ccactgagat
     2161 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2221 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2281 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2341 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2401 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2461 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2521 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2581 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2641 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2701 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2761 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2821 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2881 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2941 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3001 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3061 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     3121 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3181 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3241 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3301 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3361 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3421 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3481 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3541 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3601 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3661 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3721 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3781 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3841 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3901 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3961 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4021 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4081 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4141 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4201 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4261 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4321 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4381 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4441 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4501 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4561 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4621 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4681 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4741 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4801 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4861 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4921 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4981 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5041 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     5101 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5161 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5221 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5281 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5341 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5401 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5461 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5521 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5581 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5641 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5701 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5761 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5821 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5881 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5941 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     6001 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     6061 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     6121 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     6181 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     6241 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     6301 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     6361 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     6421 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     6481 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     6541 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6601 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     6661 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     6721 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     6781 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     6841 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     6901 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     6961 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     7021 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catg
//
