LOCUS       sequence-212929-        6702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient transfection of human USP30 C77A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6702)
  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 6702)
  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 (1-517, C77A, MG-38-16)
FEATURES             Location/Qualifiers
     source          1..6702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2005..2027
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2089..2108
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2150..2169
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2150..2168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2169..2193
                     /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)."
     promoter        2209..2318
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             3890..3907
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3997..4016)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4062..4117
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4116..4135)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4205..4252
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4265..4970
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4353..4725
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4382..4970)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4979..5012)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(5180..5767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5259..5278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5780..6571,6572..6640))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6403..6422
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg
     1021 taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct
     1081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1141 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1201 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1261 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1321 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1381 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1441 atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1501 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1561 gggggggggg cgcgcgccgg gggggggggg gggggggggg gggggggggg gggggggggg
     1621 ggggggggcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1681 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1741 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1801 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctccttcgg gctgtaatta
     1861 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1921 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc
     1981 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2041 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2101 ttattgtgct gtctcatcat tttggcaaag aattggatcg gaccgaaatt aatacgactc
     2161 actatagggg aattgtgagc ggataacaat tccccggagt taatccggga cctttaattc
     2221 aacccaacac aatatattat agttaaataa gaattattat caaatcattt gtatattaat
     2281 taaaatacta tactgtaaat tacattttat ttacaatcaa aggagatata ccatgctgag
     2341 cagccgtgcc gaagcagcaa tgaccgcagc agatcgtgca attcagcgtt ttctgcgtac
     2401 cggtgcagca gttcgttata aagttatgaa aaactggggt gtgattggtg gtattgcagc
     2461 agcactggca gcaggtattt atgttatttg gggtccgatt accgaacgta aaaaacgtcg
     2521 taaaggtctg gttccgggtc tggttaatct gggtaatacc gcttttatga atagcctgct
     2581 gcagggtctg agcgcatgtc cggcatttat tcgttggctg gaagaattca ccagccagta
     2641 tagccgtgat cagaaagaac cgcctagcca tcagtatctg agcctgaccc tgctgcatct
     2701 gctgaaagca ctgagctgtc aagaggttac cgatgatgaa gttctggatg caagctgtct
     2761 gctggatgtt ctgcgtatgt atcgttggca gattagcagc tttgaagaac aggatgcaca
     2821 tgaactgttt catgttatta ccagcagcct ggaagatgaa cgtgatcgtc agcctcgtgt
     2881 tacccacctg tttgatgttc atagcctgga acagcagagc gaaattaccc cgaaacaaat
     2941 tacctgtcgt acccgtggta gtccgcatcc gaccagcaat cattggaaaa gccagcatcc
     3001 gtttcatggt cgtctgacca gtaatatggt ttgtaaacat tgcgaacatc agagtccggt
     3061 tcgttttgat acctttgata gcctgagcct gtcaattccg gcagcaacct ggggtcatcc
     3121 gctgaccctg gatcattgtc tgcatcattt cattagcagc gaaagcgttc gtgatgttgt
     3181 ttgtgataac tgcaccaaaa ttgaagcaaa aggcaccctg aatggtgaaa aagttgaaca
     3241 tcagcgtacc acctttgtga aacagctgaa actgggtaaa ctgccgcagt gtctgtgtat
     3301 tcatctgcag cgtctgtctt ggagcagcca tggtacaccg ctgaaacgtc atgaacatgt
     3361 gcagtttaat gagttcctga tgatggacat ctataaatac catctgctgg gccataaacc
     3421 gagccagcat aatccgaaac tgaacaaaaa tccgggtccg accctggaac tgcaggatgg
     3481 tccgggtgca ccgacaccgg ttctgaatca gcctggcgca ccgaaaaccc agatttttat
     3541 gaacggtgca tgtagcccga gtctgctgcc gaccctgagt gcaccgatgc cgtttccgct
     3601 gccggttgtt ccggattata gcagcagtac ctacctgttt cgtctgatgg cagttgttgt
     3661 tcatcatggt gatatgcata gcggtcattt tgttacctat cgtcgtagcc ctccgagcgc
     3721 acgtaatccg ctgagcacca gtaatcagtg gctgtgggtt agtgatgata ccgttcgtaa
     3781 agcaagcctg caagaagttc tgagcagcag cgcatatctg ctgttttacg agcgtgttct
     3841 gagccgtatg cagcatcaga gccaagaatg taaaagcgaa gaataaaaac atcaccatca
     3901 ccatcactaa gtgattaacc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg
     3961 gccaatgccc tggctcacaa ataccactga gatcgatctt tttccctctg ccaaaaatta
     4021 tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt
     4081 cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc
     4141 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat
     4201 gtaactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagc
     4261 atgcggagga aattctcctt gaagtttccc tggtgttcaa agtaaaggag tttgcaccag
     4321 acgcacctct gttcactggt ccggcgtatt aaaacacgat acattgttat tagtacattt
     4381 attaagcgct agattctgtg cgttgttgat ttacagacaa ttgttgtacg tattttaata
     4441 attcattaaa tttataatct ttagggtggt atgttagagc gaaaatcaaa tgattttcag
     4501 cgtctttata tctgaattta aatattaaat cctcaataga tttgtaaaat aggtttcgat
     4561 tagtttcaaa caagggttgt ttttccgaac cgatggctgg actatctaat ggattttcgc
     4621 tcaacgccac aaaacttgcc aaatcttgta gcagcaatct agctttgtcg atattcgttt
     4681 gtgttttgtt ttgtaataaa ggttcgacgt cgttcaaaat attatgcgct tttgtatttc
     4741 tttcatcact gtcgttagtg tacaattgac tcgacgtaaa cacgttaaat agagcttgga
     4801 catatttaac atcgggcgtg ttagctttat taggccgatt atcgtcgtcg tcccaaccct
     4861 cgtcgttaga agttgcttcc gaagacgatt ttgccatagc cacacgacgc ctattaattg
     4921 tgtcggctaa cacgtccgcg atcaaatttg tagttgagct ttttggaatt gcgatcgcat
     4981 aacttcgtat agcatacatt atacgaagtt ataagctcgg aacgctgcgc tcggtcgttc
     5041 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5101 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5161 aggccgcgtt gctggcgttt ttccataggc tccgccccct gacgagcatc acaaaaatcg
     5221 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5281 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5341 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5401 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5461 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5521 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5581 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5641 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5701 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5761 atctcaagaa gatcctttgt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5821 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5881 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5941 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6001 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6061 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6121 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6181 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6241 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6301 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6361 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6421 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6481 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6541 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6601 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6661 attgaagcat ttatcagggt tattgtctca tgtccgcgcg tt
//
