LOCUS       sequence-261504-        5769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His6-Ubc9.
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
FEATURES             Location/Qualifiers
     source          1..5769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             42..59
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             69..86
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             576..593
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(646..664)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      660..707
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      744..1199
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(831..850)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1041..1062
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1291..2106)
                     /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(1291..2106)
                     /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     2014..2033
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2098..2117
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2228..2816
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2717..2736
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2970..2987
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3002..3141
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3067..3086)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3227..3249
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3243..3434)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4243..5325)
                     /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     5287..5306
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5326..5403)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5609..5628
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5712..5731
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5712..5730
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5731..5755
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
       61 gcagcggcct ggtgccgcgc ggcagccata tgatgtccgg aattgccctc tctcgcttag
      121 ctcaggaacg caaagcgtgg cgcaaagatc acccgtttgg gtttgttgcc gtgcctacca
      181 aaaatcctga cggtacgatg aatcttatga actgggaatg cgcgatccct ggcaagaaag
      241 gcacaccgtg ggaaggcggc ttgttcaagc tgcgtatgtt gttcaaagat gattacccgt
      301 caagtccgcc taaatgtaaa ttcgagccgc cgttatttca tccaaacgtg tatcccagcg
      361 gtacggtttg cctgagcatc cttgaagaag acaaagattg gcgtcccgca attaccatca
      421 agcagattct gctcggcatt caagagctgt taaatgaacc aaacattcag gacccagcgc
      481 aggcagaagc ctatactatc tactgtcaga accgtgtaga gtatgagaag cgcgtccgtg
      541 cacaagcgaa aaagtttgcc ccgtcgtaac tcgagcacca ccaccaccac cactgagatc
      601 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
      661 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
      721 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
      781 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      841 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
      901 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
      961 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1021 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1081 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1141 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     1201 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1261 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     1321 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     1381 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     1441 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     1501 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     1561 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     1621 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     1681 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     1741 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     1801 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     1861 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     1921 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     1981 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     2041 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     2101 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     2161 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2221 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2281 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2341 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2401 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2461 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2521 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2581 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2641 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2701 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2761 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2821 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2881 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2941 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3001 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     3061 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3121 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3181 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3241 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3301 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3361 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3421 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     3481 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3541 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     3601 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     3661 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     3721 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     3781 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     3841 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     3901 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     3961 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4021 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     4081 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     4141 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4201 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4261 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4321 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4381 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     4441 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     4501 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4561 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     4621 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     4681 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     4741 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     4801 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     4861 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     4921 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     4981 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5041 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     5101 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5161 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5221 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     5281 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5341 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     5401 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5461 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     5521 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     5581 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     5641 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     5701 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     5761 agaaataat
//
