LOCUS       sequence-212899-        7007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human USP30 (construct 13).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7007)
  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 7007)
  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 13, MG-26-82)
FEATURES             Location/Qualifiers
     source          1..7007
                     /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             2080..2097
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2164..2211
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2248..2703
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2335..2354)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2545..2566
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2795..3610)
                     /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(2795..3610)
                     /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     3518..3537
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3602..3621
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3732..4320
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4221..4240
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4474..4491
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4506..4645
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4571..4590)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4731..4753
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4747..4938)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5747..6829)
                     /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     6791..6810
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6830..6907)
                     /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 gaacgtgatg gttccggctc tcattggaaa agccagcatc cgtttcatgg
     1441 tcgtctgacc agtaatatgg tttgtaaaca ttgcgaacat cagagtccgg ttcgttttga
     1501 tacctttgat agcctgagcc tgtcaattcc ggcagcaacc tggggtcatc cgctgaccct
     1561 ggatcattgt ctgcatcatt tcattagcag cgaaagcgtt cgtgatgttg tttgtgataa
     1621 ctgcaccaaa attgaagcaa aaggcaccct gaatggtgaa aaagttgaac atcagcgtac
     1681 cacctttgtg aaacagctga aactgggtaa actgccgcag tgtctgtgta ttcatctgca
     1741 gcgtctgtct tggagcagcc atggtacacc gctgaaacgt catgaacatg tgcagtttaa
     1801 tgaggacctg agcatggacg agtataaata ccactccaat gcgagtacct acctgtttcg
     1861 tctgatggca gttgttgttc atcatggtga tatgcatagc ggtcattttg ttacctatcg
     1921 tcgtagccct ccgagcgcac gtaatccgct gagcaccagt aatcagtggc tgtgggttag
     1981 tgatgatacc gttcgtaaag caagcctgca agaagttctg agcagcagcg catatctgct
     2041 gttttacgag cgtgtttaaa gctttctaga ccatttaaac accaccacca ccaccactga
     2101 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     2161 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     2221 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     2281 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2341 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2401 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2461 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2521 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2581 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2641 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2701 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2761 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     2821 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     2881 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     2941 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     3001 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     3061 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     3121 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     3181 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     3241 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     3301 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     3361 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     3421 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     3481 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     3541 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     3601 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     3661 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3721 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3781 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3841 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3901 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3961 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4021 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4081 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4141 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4201 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4261 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4321 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4381 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4441 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4501 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     4561 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     4621 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     4681 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4741 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     4801 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     4861 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     4921 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     4981 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5041 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5101 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     5161 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     5221 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     5281 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     5341 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     5401 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     5461 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     5521 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     5581 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     5641 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     5701 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     5761 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5821 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     5881 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     5941 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     6001 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     6061 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     6121 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     6181 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     6241 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     6301 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     6361 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     6421 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     6481 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     6541 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     6601 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     6661 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     6721 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     6781 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     6841 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     6901 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     6961 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatg
//
