LOCUS       sequence-261491-        5769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His6-Ubc9-C93A.
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-C93A
FEATURES             Location/Qualifiers
     source          1..5769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             19..36
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             46..63
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             553..570
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(623..641)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      637..684
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      721..1176
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(808..827)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1018..1039
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1268..2083)
                     /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(1268..2083)
                     /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     1991..2010
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2075..2094
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2205..2793
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2694..2713
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2947..2964
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2979..3118
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3044..3063)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3204..3226
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3220..3411)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4220..5302)
                     /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     5264..5283
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5303..5380)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5586..5605
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5689..5708
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5689..5707
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5708..5732
                     /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             5747..5769
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tataccatgg gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc
       61 agccatatga tgtccggaat tgccctctct cgcttagctc aggaacgcaa agcgtggcgc
      121 aaagatcacc cgtttgggtt tgttgccgtg cctaccaaaa atcctgacgg tacgatgaat
      181 cttatgaact gggaatgcgc gatccctggc aagaaaggca caccgtggga aggcggcttg
      241 ttcaagctgc gtatgttgtt caaagatgat tacccgtcaa gtccgcctaa atgtaaattc
      301 gagccgccgt tatttcatcc aaacgtgtat cccagcggta cggttgccct gagcatcctt
      361 gaagaagaca aagattggcg tcccgcaatt accatcaagc agattctgct cggcattcaa
      421 gagctgttaa atgaaccaaa cattcaggac ccagcgcagg cagaagccta tactatctac
      481 tgtcagaacc gtgtagagta tgagaagcgc gtccgtgcac aagcgaaaaa gtttgccccg
      541 tcgtaactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
      601 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
      661 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
      721 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      781 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      841 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      901 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      961 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1021 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1081 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1141 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     1201 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     1261 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     1321 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     1381 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     1441 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     1501 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     1561 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     1621 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     1681 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     1741 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     1801 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     1861 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     1921 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     1981 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     2041 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     2101 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     2161 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2221 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2281 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2341 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2401 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2461 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     2521 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     2581 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     2641 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2701 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2761 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2821 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     2881 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     2941 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3001 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     3061 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3121 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3181 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3241 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     3301 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     3361 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     3421 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     3481 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     3541 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     3601 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     3661 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     3721 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     3781 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     3841 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     3901 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     3961 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     4021 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     4081 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     4141 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     4201 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4261 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     4321 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     4381 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     4441 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     4501 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     4561 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     4621 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     4681 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     4741 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     4801 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     4861 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     4921 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     4981 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     5041 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     5101 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     5161 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     5221 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     5281 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     5341 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     5401 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     5461 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     5521 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     5581 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     5641 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     5701 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     5761 agaaggaga
//
