LOCUS       sequence-366793-        5769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His6-Ubc9-K14R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5769)
  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 5769)
  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: pET28-His6-Ubc9-K14R
FEATURES             Location/Qualifiers
     source          1..5769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(413..1495)
                     /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     1457..1476
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1496..1573)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1779..1798
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1882..1901
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1882..1900
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1901..1925
                     /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             1940..1962
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1981..1998
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2008..2025
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2515..2532
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2585..2603)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2599..2646
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2683..3138
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2770..2789)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2980..3001
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3230..4045)
                     /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(3230..4045)
                     /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     3953..3972
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4037..4056
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4167..4755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4656..4675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4909..4926
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4941..5080
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5006..5025)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5166..5188
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5182..5373)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
       61 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
      121 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
      181 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
      241 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
      301 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
      361 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
      421 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
      481 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
      541 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
      601 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
      661 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
      721 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
      781 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
      841 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
      901 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
      961 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     1021 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     1081 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     1141 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     1201 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     1261 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     1321 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     1381 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     1441 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     1501 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     1561 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     1621 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     1681 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     1741 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     1801 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     1861 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     1921 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc
     1981 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat gatgtccgga
     2041 attgccctct ctcgcttagc tcaggaacgc cgtgcgtggc gcaaagatca cccgtttggg
     2101 tttgttgccg tgcctaccaa aaatcctgac ggtacgatga atcttatgaa ctgggaatgc
     2161 gcgatccctg gcaagaaagg cacaccgtgg gaaggcggct tgttcaagct gcgtatgttg
     2221 ttcaaagatg attacccgtc aagtccgcct aaatgtaaat tcgagccgcc gttatttcat
     2281 ccaaacgtgt atcccagcgg tacggtttgc ctgagcatcc ttgaagaaga caaagattgg
     2341 cgtcccgcaa ttaccatcaa gcagattctg ctcggcattc aagagctgtt aaatgaacca
     2401 aacattcagg acccagcgca ggcagaagcc tatactatct actgtcagaa ccgtgtagag
     2461 tatgagaagc gcgtccgtgc acaagcgaaa aagtttgccc cgtcgtaact cgagcaccac
     2521 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     2581 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     2641 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     2701 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2761 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2821 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2881 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2941 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3001 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3061 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3121 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3181 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     3241 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     3301 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     3361 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     3421 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     3481 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     3541 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     3601 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     3661 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     3721 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3781 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3841 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3901 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3961 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     4021 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     4081 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4141 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4201 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4261 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4321 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4381 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4441 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4501 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4561 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4621 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4681 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4741 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4801 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4861 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4921 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4981 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     5041 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     5101 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5161 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     5221 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     5281 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     5341 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     5401 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     5461 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     5521 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     5581 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     5641 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     5701 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     5761 ttcattctg
//
