LOCUS       sequence-261488-        5769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His6-Ubc9-C138A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5769)
  AUTHORS   Hofmann R, Akimoto G, Wucherpfennig TG, Zeymer C, Bode JW
  TITLE     Lysine acylation using conjugating enzymes for site-specific 
            modification and ubiquitination of recombinant proteins.
  JOURNAL   Nat Chem. 2020 Nov;12(11):1008-1015. doi: 10.1038/s41557-020-0528-y.
            Epub 2020 Sep 14.
  PUBMED    32929246
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: 
            "10.1038/s41557-020-0528-y"; journalName: "Nat Chem"; date: 
            "2020-11"; volume: "12"; issue: "11"; pages: "1008-1015"
COMMENT     Sequence Label: pET28-His6-Ubc9-C138A
FEATURES             Location/Qualifiers
     source          1..5769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             331..348
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(401..419)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      415..462
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      499..954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(586..605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     796..817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1046..1861)
                     /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(1046..1861)
                     /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     1769..1788
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1853..1872
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1983..2571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2472..2491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2725..2742
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2757..2896
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2822..2841)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2982..3004
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2998..3189)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3998..5080)
                     /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     5042..5061
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5081..5158)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5364..5383
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5467..5486
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5467..5485
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5486..5510
                     /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             5525..5547
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5566..5583
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5593..5610
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 ccgtgggaag gcggcttgtt caagctgcgt atgttgttca aagatgatta cccgtcaagt
       61 ccgcctaaat gtaaattcga gccgccgtta tttcatccaa acgtgtatcc cagcggtacg
      121 gtttgcctga gcatccttga agaagacaaa gattggcgtc ccgcaattac catcaagcag
      181 attctgctcg gcattcaaga gctgttaaat gaaccaaaca ttcaggaccc agcgcaggca
      241 gaagcctata ctatctacgc gcagaaccgt gtagagtatg agaagcgcgt ccgtgcacaa
      301 gcgaaaaagt ttgccccgtc gtaactcgag caccaccacc accaccactg agatccggct
      361 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      421 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      481 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      541 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      601 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      661 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
      721 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
      781 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
      841 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
      901 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
      961 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1021 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     1081 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     1141 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     1201 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     1261 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     1321 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     1381 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     1441 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     1501 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     1561 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     1621 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     1681 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     1741 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     1801 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     1861 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     1921 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1981 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2041 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2101 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2161 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2221 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2281 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2341 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2401 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2461 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2521 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2581 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2641 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2701 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     2761 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     2821 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     2881 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     2941 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3001 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     3061 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     3121 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     3181 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     3241 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     3301 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     3361 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     3421 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     3481 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     3541 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     3601 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     3661 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3721 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3781 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3841 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     3901 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     3961 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4021 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4081 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     4141 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     4201 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     4261 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     4321 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     4381 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     4441 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     4501 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     4561 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     4621 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     4681 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     4741 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     4801 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     4861 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     4921 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     4981 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     5041 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     5101 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     5161 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     5221 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     5281 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     5341 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     5401 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     5461 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     5521 taattttgtt taactttaag aaggagatat accatgggca gcagccatca tcatcatcat
     5581 cacagcagcg gcctggtgcc gcgcggcagc catatgatgt ccggaattgc cctctctcgc
     5641 ttagctcagg aacgcaaagc gtggcgcaaa gatcacccgt ttgggtttgt tgccgtgcct
     5701 accaaaaatc ctgacggtac gatgaatctt atgaactggg aatgcgcgat ccctggcaag
     5761 aaaggcaca
//
