LOCUS       sequence-458328-        8618 bp    DNA     circular SYN 15-APR-2025
DEFINITION  E547-mutant of human USP7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8618)
  AUTHORS   Bezsonova Lab
  TITLE     Functional Spectrum of USP7 Pathogenic Hao-Fountain Syndrome: 
            Insights into the Enzyme's Activity, Stability and Allosteric 
            Modulation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8618)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: E547* FL USP7
FEATURES             Location/Qualifiers
     source          1..8618
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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             3543..3565
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3584..3601
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3611..3628
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6967..6984
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7037..7055)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7051..7098
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7135..7590
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7222..7241)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7432..7453
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7682..8497)
                     /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(7682..8497)
                     /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     8405..8424
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8489..8508
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3541 attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca
     3601 cagcagcggc ctggttccgc gtggtagtat gaaccaccag cagcagcagc agcagcagaa
     3661 agcgggcgag cagcagttga gcgagcccga ggacatggag atggaagcgg gagatacaga
     3721 tgacccacca agaattactc agaaccctgt gatcaatggg aatgtggccc tgagtgatgg
     3781 acacaacacc gcggaggagg acatggagga tgacaccagt tggcgctccg aggcaacctt
     3841 tcagttcact gtggagcgct tcagcagact gagtgagtcg gtccttagcc ctccgtgttt
     3901 tgtgcgaaat ctgccatgga agattatggt gatgccacgc ttttatccag acagaccaca
     3961 ccaaaaaagc gtaggattct ttctccagtg caatgctgaa tctgattcca cgtcatggtc
     4021 ttgccatgca caagcagtgc tgaagataat aaattacaga gatgatgaaa agtcgttcag
     4081 tcgtcgtatt agtcatttgt tcttccataa agaaaatgat tggggatttt ccaattttat
     4141 ggcctggagt gaagtgaccg atcctgagaa aggatttata gatgatgaca aagttacctt
     4201 tgaagtcttt gtacaggcgg atgctcccca tggagttgcg tgggattcaa agaagcacac
     4261 aggctacgtc ggcttaaaga atcagggagc gacttgttac atgaacagcc tgctacagac
     4321 gttatttttc acgaatcagc tacgaaaggc tgtgtacatg atgccaaccg agggggatga
     4381 ttcgtctaaa agcgtccctt tagcattaca aagagtgttc tatgaattac agcatagtga
     4441 taaacctgta ggaacaaaaa agttaacaaa gtcatttggg tgggaaactt tagatagctt
     4501 catgcaacat gatgttcagg agctttgtcg agtgttgctc gataatgtgg aaaataagat
     4561 gaaaggcacc tgtgtagagg gcaccatacc caaattattc cgcggcaaaa tggtgtccta
     4621 tatccagtgt aaagaagtag actatcggtc tgatagaaga gaagattatt atgatatcca
     4681 gctaagtatc aaaggaaaga aaaatatatt tgaatcattt gtggattatg tggcagtaga
     4741 acagctcgat ggggacaata aatacgacgc tggggaacat ggcttacagg aagcagagaa
     4801 aggtgtgaaa ttcctaacat tgccaccagt gttacatcta caactgatga gatttatgta
     4861 tgaccctcag acggaccaaa atatcaagat caatgatagg tttgaattcc cagagcagtt
     4921 accacttgat gaatttttgc aaaaaacaga tcctaaggac cctgcaaatt atattcttca
     4981 tgcagtcctg gttcatagtg gagataatca tggtggacat tatgtggttt atctaaaccc
     5041 caaaggggat ggcaaatggt gtaaatttga tgacgacgtg gtgtcaaggt gtactaaaga
     5101 ggaagcaatt gagcacaatt atgggggtca cgatgacgac ctgtctgttc gacactgcac
     5161 taatgcttac atgttagtct acatcaggga atcaaaactg agtgaagttt tacaggcggt
     5221 caccgaccat gatattcctc agcagttggt ggagcgatta caagaataga aaaggatcga
     5281 ggctcagaag cggaaggagc ggcaggaagc ccatctctat atgcaagtgc agatagtcgc
     5341 agaggaccag ttttgtggcc accaagggaa tgacatgtac gatgaagaaa aagtgaaata
     5401 cactgtgttc aaagtattga agaactcctc gcttgctgag tttgttcaga gcctctctca
     5461 gaccatggga tttccacaag atcaaattcg attgtggccc atgcaagcaa ggagtaatgg
     5521 aacaaaacga ccagcaatgt tagataatga agccgacggc aataaaacaa tgattgagct
     5581 cagtgataat gaaaaccctt ggacaatatt cctggaaaca gttgatcccg agctggctgc
     5641 tagtggagcg accttaccca agtttgataa agatcatgat gtaatgttat ttttgaagat
     5701 gtatgatccc aaaacgcgga gcttgaatta ctgtgggcat atctacacac caatatcctg
     5761 taaaatacgt gacttgctcc cagttatgtg tgacagagca ggatttattc aagatactag
     5821 ccttatcctc tatgaggaag ttaaaccgaa tttaacagag agaattcagg actatgacgt
     5881 gtctcttgat aaagcccttg atgaactaat ggatggtgac atcatagtat ttcagaagga
     5941 tgaccctgaa aatgataaca gtgaattacc caccgcaaag gagtatttcc gagatctcta
     6001 ccaccgcgtt gatgtcattt tctgtgataa aacaatccct aatgatcctg gatttgtggt
     6061 tacgttatca aatagaatga attattttca ggttgcaaag acagttgcac agaggctcaa
     6121 cacagatcca atgttgctgc agtttttcaa gtctcaaggt tatagggatg gcccaggtaa
     6181 tcctcttaga cataattatg aaggtacttt aagagatctt ctacagttct tcaagcctag
     6241 acaacctaag aaactttact atcagcagct taagatgaaa atcacagact ttgagaacag
     6301 gcgaagtttt aaatgtatat ggttaaacag ccaatttagg gaagaggaaa taacactata
     6361 tccagacaag catgggtgtg tccgggacct gttagaagaa tgtaaaaagg ccgtggagct
     6421 tggggagaaa gcatcaggga aacttaggct gctagaaatt gtaagctaca aaatcattgg
     6481 tgttcatcaa gaagatgaac tattagaatg tttatctcct gcaacgagcc ggacgtttcg
     6541 aatagaggaa atccctttgg accaggtgga catagacaaa gagaatgaga tgcttgtcac
     6601 agtggcgcat ttccacaaag aggtcttcgg aacgttcgga atcccgtttt tgctgaggat
     6661 acaccagggc gagcattttc gagaagtgat gaagcgaatc cagagcctgc tggacatcca
     6721 ggagaaggag tttgagaagt ttaaatttgc aattgtaatg atgggccgac accagtacat
     6781 aaatgaagac gagtatgaag taaatttgaa agactttgag ccacagcccg gtaatatgtc
     6841 tcatcctcgg ccttggctag ggctcgacca cttcaacaaa gccccaaaga ggagtcgcta
     6901 cacttacctt gaaaaggcca ttaaaatcca taactgatga tgacgaagct tgcggccgca
     6961 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     7021 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     7081 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     7141 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     7201 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     7261 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     7321 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     7381 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     7441 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     7501 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     7561 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     7621 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     7681 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     7741 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     7801 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     7861 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     7921 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     7981 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     8041 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     8101 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     8161 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     8221 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     8281 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     8341 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     8401 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     8461 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     8521 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     8581 tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
