LOCUS       sequence-212891-        7322 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human USP30 (construct 2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7322)
  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 7322)
  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 2, MG-26-18)
FEATURES             Location/Qualifiers
     source          1..7322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     55..74
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        55..73
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    74..98
                     /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             113..135
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             154..171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             181..204
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             205..858
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             877..900
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2241..2258
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2325..2372
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2409..2864
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2496..2515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2706..2727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2956..3771)
                     /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(2956..3771)
                     /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     3679..3698
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3763..3782
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3893..4481
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4382..4401
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4635..4652
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4667..4806
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4732..4751)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4892..4914
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4908..5099)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5908..6990)
                     /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     6952..6971
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6991..7068)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7274..7293
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
       61 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
      121 actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggt
      181 ctggaagttc tgtttcaggg cccgatgtcc cctatactag gttattggaa aattaagggc
      241 cttgtgcaac ccactcgact tcttttggaa tatcttgaag aaaaatatga agagcatttg
      301 tatgagcgcg atgaaggtga taaatggcga aacaaaaagt ttgaattggg tttggagttt
      361 cccaatcttc cttattatat tgatggtgat gttaaattaa cacagtctat ggccatcata
      421 cgttatatag ctgacaagca caacatgttg ggtggttgtc caaaagagcg tgcagagatt
      481 tcaatgcttg aaggagcggt tttggatatt agatacggtg tttcgagaat tgcatatagt
      541 aaagactttg aaactctcaa agttgatttt cttagcaagc tacctgaaat gctgaaaatg
      601 ttcgaagatc gtttatgtca taaaacatat ttaaatggtg atcatgtaac ccatcctgac
      661 ttcatgttgt atgacgctct tgatgttgtt ttatacatgg acccaatgtg cctggatgcg
      721 ttcccaaaat tagtttgttt taaaaaacgt attgaagcta tcccacaaat tgataagtac
      781 ttgaaatcca gcaagtatat agcatggcct ttgcagggct ggcaagccac gtttggtggt
      841 ggcgaccatc ctccaaaatc ggatctgagc agcggtctgg aagttctgtt tcagggcccg
      901 aaaggtctgg ttccgggtct ggttaatctg ggtaatacct gttttatgaa tagcctgctg
      961 cagggtctga gcgcatgtcc ggcatttatt cgttggctgg aagaattcac cagccagtat
     1021 agccgtgatc agaaagaacc gcctagccat cagtatctga gcctgaccct gctgcatctg
     1081 ctgaaagcac tgagctgtca agaggttacc gatgatgaag ttctggatgc aagctgtctg
     1141 ctggatgttc tgcgtatgta tcgttggcag attagcagct ttgaagaaca ggatgcacat
     1201 gaactgtttc atgttattac cagcagcctg gaagatgaac gtgatcgtca gcctcgtgtt
     1261 acccacctgt ttgatgttca tagcctggaa cagcagagcg aaattacccc gaaacaaatt
     1321 acctgtcgta cccgtggtag tccgcatccg accagcaatc attggaaaag ccagcatccg
     1381 tttcatggtc gtctgaccag taatatggtt tgtaaacatt gcgaacatca gagtccggtt
     1441 cgttttgata cctttgatag cctgagcctg tcaattccgg cagcaacctg gggtcatccg
     1501 ctgaccctgg atcattgtct gcatcatttc attagcagcg aaagcgttcg tgatgttgtt
     1561 tgtgataact gcaccaaaat tgaagcaaaa ggcaccctga atggtgaaaa agttgaacat
     1621 cagcgtacca cctttgtgaa acagctgaaa ctgggtaaac tgccgcagtg tctgtgtatt
     1681 catctgcagc gtctgtcttg gagcagccat ggtacaccgc tgaaacgtca tgaacatgtg
     1741 cagtttaatg agttcctgat gatggacatc tataaatacc atctgctggg ccataaaccg
     1801 agccagcata atccgaaact gaacaaaaat ccgggtccga ccctggaact gcaggatggt
     1861 ccgggtgcac cgacaccggt tctgaatcag cctggcgcac cgaaaaccca gatttttatg
     1921 aacggtgcat gtagcccgag tctgctgccg accctgagtg caccgatgcc gtttccgctg
     1981 ccggttgttc cggattatag cagcagtacc tacctgtttc gtctgatggc agttgttgtt
     2041 catcatggtg atatgcatag cggtcatttt gttacctatc gtcgtagccc tccgagcgca
     2101 cgtaatccgc tgagcaccag taatcagtgg ctgtgggtta gtgatgatac cgttcgtaaa
     2161 gcaagcctgc aagaagttct gagcagcagc gcatatctgc tgttttacga gcgtgtttaa
     2221 agctttctag accatttaaa caccaccacc accaccactg agatccggct gctaacaaag
     2281 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     2341 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     2401 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2461 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2521 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2581 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2641 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2701 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2761 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2821 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     2881 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2941 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     3001 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     3061 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     3121 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     3181 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     3241 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     3301 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     3361 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     3421 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     3481 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     3541 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     3601 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     3661 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     3721 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     3781 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     3841 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3901 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3961 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4021 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4081 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4141 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4201 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4261 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4321 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4381 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4441 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4501 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4561 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4621 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     4681 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     4741 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4801 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4861 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4921 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     4981 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     5041 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     5101 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     5161 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     5221 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     5281 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     5341 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     5401 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     5461 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     5521 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     5581 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     5641 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     5701 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     5761 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     5821 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     5881 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5941 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6001 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     6061 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     6121 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     6181 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     6241 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     6301 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     6361 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     6421 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     6481 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     6541 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     6601 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     6661 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     6721 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6781 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6841 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6901 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6961 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     7021 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     7081 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     7141 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     7201 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     7261 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     7321 tg
//
