LOCUS       sequence-370408-        7844 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His-Ub, Ubc9 and ChE1 v4.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7844)
  AUTHORS   Akimoto G, Fernandes AP, Bode JW
  TITLE     Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric 
            E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9.
  JOURNAL   ACS Cent Sci. 2022 Feb 23;8(2):275-281. doi: 
            10.1021/acscentsci.1c01490. Epub 2022 Feb 9.
  PUBMED    35237717
REFERENCE   2  (bases 1 to 7844)
  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"; doi: 
            "10.1021/acscentsci.1c01490"; journalName: "ACS Cent Sci"; date: 
            "2022-02-23- 23"; volume: "8"; issue: "2"; pages: "275-281"
COMMENT     Sequence Label: pUb-HisUb-Ubc9-ChE1 v4.5
FEATURES             Location/Qualifiers
     source          1..7844
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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)."
     CDS             101..118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             140..160
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             173..400
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     promoter        549..567
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    568..592
                     /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)."
     CDS             1124..1168
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      1220..1267
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1352..1370
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1371..1395
                     /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)."
     terminator      4543..4590
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4761..5552)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(5553..5644)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5692..6430)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(6640..7722)
                     /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     7684..7703
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7723..7800)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctgt agaaataatt
       61 ttgtttaact ttaataagga gatataccat gggcagcagc catcaccatc atcaccacga
      121 ttacgatatc ccaacgaccg aaaacctgta ttttcagggc gccatgggat ccatgcaaat
      181 tttcgtaaag accctgaccg ggaagaccat tacacttgag gtggagccca gcgacacaat
      241 tgagaacgtc aaggccaaga tccaggataa agagggcatc ccgcctgacc aacaacgttt
      301 aatcttcgca ggcaagcagt tagaggacgg ccgtacactg agtgactata atatccaaaa
      361 ggaatctacc ctgcacttgg ttctccgcct tcgtggtggc tgaggatccc cgggaattca
      421 tcgtgactga ctgacgctgc aggtcgacaa gcttgcggcc gcataatgct taaccccttg
      481 gggcctctaa acgggtcttg aggggttttt tgcatgcatc gtattgtaca cggccgcata
      541 atcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt ccccatctta
      601 gtatattagt taagtataag aaggagatat acatatgtcc ggaattgccc tctctcgctt
      661 agctcaggaa cgccgtgcgt ggcgcaaaga tcacccgttt gggtttgttg ccgtgcctac
      721 caaaaatcct gacggtacga tgaatcttat gaactgggaa tgcgcgatcc ctggcaagaa
      781 aggcacaccg tgggaaggcg gcttgttcaa gctgcgtatg ttgttcaaag atgattaccc
      841 gtcaagtccg cctaaatgta aattcgagcc gccgttattt catccaaacg tgtatcccag
      901 cggtacggtt tgcctgagca tccttgaaga agacaaagat tggcgtcccg caattaccat
      961 caagcagatt ctgctcggca ttcaagagct gttaaatgaa ccaaacattc aggacccagc
     1021 gcaggcagaa gcctatacta tctactgtca gaaccgtgta gagtatgaga agcgcgtccg
     1081 tgcacaagcg aaaaagtttg ccccgtcgta actcgagtct ggtaaagaaa ccgctgctgc
     1141 gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa cctaggctgc
     1201 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1261 ttttttgctg aaacctcagg catttgagaa gcacggtacc attaagtcga acagaaagta
     1321 atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg ggaattgtga
     1381 gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga tatacatatg
     1441 gccaagaacg gcagtgaagc agacatagac gagggccttt actcccggca gctgtatgtg
     1501 ttgggccatg aggcaatgaa gcggctccag acatccagtg tcctggtatc aggcctgcgg
     1561 ggcctgggcg tggagatcgc taagaacatc atccttggtg gggtcaaggc tgttacccta
     1621 catgaccagg gcactgccca gtgggctgat ctttcctccc agttctacct gcgggaggag
     1681 gacatcggta aaaaccgggc cgaggtatca cagccccgcc tcgctgagct caacagctat
     1741 gtgcctgtca ctgcctacac tggacccctc gttgaggact tccttagtgg tttccaggtg
     1801 gtggtgctca ccaacacccc cctggaggac cagctgcgag tgggtgagtt ctgtcacaac
     1861 cgtggcatca agctggtggt ggcagacacg cggggcctgt ttgggcaact cttctgtgac
     1921 tttggagagg aaatgatcct cacagattcc aatggggagc agccactcag tgctatggtt
     1981 tctatggtta ccaaggacaa ccccggtgtg gttacctgcc tggatgaggc ccgacacggg
     2041 tttgagagcg gggactttgt ctccttttca gaagtacagg gcatggttga actcaacgga
     2101 aatcagccca tggagatcaa agtcctgggt ccttatacct ttagcatctg tgacacctcc
     2161 aacttctccg actacatccg tggaggcatc gtcagtcagg tcaaagtacc taagaagatt
     2221 agctttaaat ccttggtggc ctcactggca gaacctgact ttgtggtgac ggacttcgcc
     2281 aagttttctc gccctgccca gctgcacatt ggcttccagg ccctgcacca gttctgtgct
     2341 cagcatggcc ggccaccccg gccccgcaat gaggaggatg cagcagaact ggtagcctta
     2401 gcacaggctg tgaatgctcg agccctgcca gcagtgcagc aaaataacct ggacgaggac
     2461 ctcatccgga agctggcata tgtggctact ggggatctgg cacccataaa cgccttcatt
     2521 gggggcctgg ctgcccagga agtcatgaag gcctgctccg ggaagttcat gcccatcatg
     2581 cagtggctat actttgatgc ccttgagtgt ctccctgtcg acaaagaggt cctcacagaa
     2641 gacaagtgcc tccagcgcca gaaccgttat gacgggcaag tggctgtgtt tggctcagac
     2701 ctgcaagaga agctgggcaa gcagaagtat ttcctggtgg gtgcgggggc cattggctgt
     2761 gagctgctca agaactttgc catgattggg ctgggctgcg gggagggtgg agaaatcatc
     2821 gttacagaca tggacaccat tgagaagtca aatctgaatc gacagtttct tttccggccc
     2881 tgggatgtca cgaagttaaa gtctgacacg gctgctgcag ctgtgcgcca aatgaatcca
     2941 catatccggg tgacaagcca ccagaaccgt gtgggtcctg acacggagcg catctatgat
     3001 gacgattttt tccaaaacct agatggcgtg gccaatgccc tggacaacgt ggatgcccgc
     3061 atgtatatgg accgccgctg tgtctactac cggaagccac tgctggagtc aggcacactg
     3121 ggtaccaaag gcaatgtgca ggtggtgatc cccttcctga cagagtcgta cagttccagc
     3181 caggacccac ctgagaagtc catccccatc tgtaccctga agaacttccc taatgccatc
     3241 gagcacaccc tgcagtgggc tcgggatgag tttgaaggcc tcttcaagca gccagcagaa
     3301 aatgtcaacc agtacctcac agaccccaag tttgtggagc gaacactgcg gctgacaggc
     3361 actcaaccct tggaggtgct ggaggccgtg cagcgcagcc tggtgctgca gcgaccacag
     3421 acctgggctg actgcgtgac ctgggcctgc caccactggc acacccagta ctcgaacaac
     3481 atccggcagc tgctgcacta catccctcct gaccagctca caagctcggg agcgccgttc
     3541 tggtctgggc ccaaacgctg tccacacccg ctcacctttg atgtcaacaa tcccctgcat
     3601 ctggactatg tgatggctgc tgccaacctg tttgcccaga cttacgggct gacaggctct
     3661 caggaccgag ctgctgtgtc cacattcctg cagtctgtgc aggtccccga atttaccccc
     3721 aagtctggcg tcaagatcca tgtttctgac caggagctgc agagcgccaa tgcctctgtt
     3781 gatgacagtc gtcttgagga gctcaaagcc actctgccca gcccagacaa gctccctgga
     3841 ttcaagatgt accccattga ctttgagaag gatgaagaca gcaactttca tatggatttc
     3901 atcgaggctg catccaacct ccgggcagaa aactatgaca ttccttctgc agaccggcac
     3961 aagagcaagc tgattgcagg gaagatcatc cccgccattg ccacgacctc agcagccgtg
     4021 gttggccttg tgtgtctgga actgtataag gttgtgcagg ggcaccgaca gcttgactcc
     4081 tacaagaatg gtttcctcaa cttggccctg cctttctttg gtttctctga accccttgcc
     4141 gcaccacgtc accagtacta taaccaagag tggacattgt gggatcgcgt gactgtgcgg
     4201 ctgaatgtac ataaagtgac tgttctcacc ttacaagaca agatagtgaa agaaaaattt
     4261 gctatggtag caccagatgt ccaaattgaa gatgggaaag gaacaatcct aatatcttcc
     4321 gaagagggag agacggaagc taataatcac aagaagttgt cagaatttgg aattagaaat
     4381 ggcagccggc ttcaagcaga tgacttcctc caggactata ctttattgat caacatcctt
     4441 catagtgaag accttggaaa ggacgttgaa tttgaagttg ttggtgatta acctagggac
     4501 cttggcccca agggctaggc tgctgccacc gctgagcaat aactagcata accccttggg
     4561 gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga gaagcacacg
     4621 gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt
     4681 taacgaccct gccctgaacc gacgaccggg tcatcgtggc cggatcttgc ggcccctcgg
     4741 cttgaacgaa ttgttagaca ttatttgccg actaccttgg tgatctcgcc tttcacgtag
     4801 tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga
     4861 taagcctgtc tagcttcaag tatgacgggc tgatactggg ccggcaggcg ctccattgcc
     4921 cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg
     4981 gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga gttccatagc
     5041 gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc aaagagttcc
     5101 tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag caagatagcc
     5161 agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt gcgctgccat
     5221 tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc gtcgtgcaca
     5281 acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc cgaagtttcc
     5341 aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc
     5401 agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt
     5461 acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc tgatagttga
     5521 gtcgatactt cggcgatcac cgcttccctc atactcttcc tttttcaata ttattgaagc
     5581 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5641 caaatagcta gctcactcgg tcgctacgct ccgggcgtga gactgcggcg ggcgctgcgg
     5701 acacatacaa agttacccac agattccgtg gataagcagg ggactaacat gtgaggcaaa
     5761 acagcagggc cgcgccggtg gcgtttttcc ataggctccg ccctcctgcc agagttcaca
     5821 taaacagacg cttttccggt gcatctgtgg gagccgtgag gctcaaccat gaatctgaca
     5881 gtacgggcga aacccgacag gacttaaaga tccccaccgt ttccggcggg tcgctccctc
     5941 ttgcgctctc ctgttccgac cctgccgttt accggatacc tgttccgcct ttctccctta
     6001 cgggaagtgt ggcgctttct catagctcac acactggtat ctcggctcgg tgtaggtcgt
     6061 tcgctccaag ctgggctgta agcaagaact ccccgttcag cccgactgct gcgccttatc
     6121 cggtaactgt tcacttgagt ccaacccgga aaagcacggt aaaacgccac tggcagcagc
     6181 cattggtaac tgggagttcg cagaggattt gtttagctaa acacgcggtt gctcttgaag
     6241 tgtgcgccaa agtccggcta cactggaagg acagatttgg ttgctgtgct ctgcgaaagc
     6301 cagttaccac ggttaagcag ttccccaact gacttaacct tcgatcaaac cacctcccca
     6361 ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat ctcaagaaga
     6421 tcctttgatc ttttctactg aaccgctcta gatttcagtg caatttatct cttcaaatgt
     6481 agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc
     6541 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     6601 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6661 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6721 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     6781 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     6841 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     6901 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     6961 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     7021 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     7081 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     7141 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     7201 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     7261 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     7321 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     7381 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     7441 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     7501 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     7561 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     7621 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     7681 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     7741 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     7801 cgggatctcg acgctctccc ttatgcgact cctgcattag gaaa
//
