LOCUS       sequence-275576-        6413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GST tagged AO7 encoding aa 126-258 with an internal deletion that 
            results in loss of E2 binding for expression in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6413)
  AUTHORS   Li S, Liang YH, Mariano J, Metzger MB, Stringer DK, Hristova VA, Li 
            J, Randazzo PA, Tsai YC, Ji X, Weissman AM
  TITLE     Insights into Ubiquitination from the Unique Clamp-like Binding of 
            the RING E3 AO7 to the E2 UbcH5B.
  JOURNAL   J Biol Chem. 2015 Dec 18;290(51):30225-39. doi: 
            10.1074/jbc.M115.685867. Epub 2015 Oct 16.
  PUBMED    26475854
REFERENCE   2  (bases 1 to 6413)
  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: 
            "10.1074/jbc.M115.685867"; journalName: "J Biol Chem"; date: 
            "2015-12-18- 18"; volume: "290"; issue: "51"; pages: "30225-39"
COMMENT     Sequence Label: pETDUET-GST AO7RE ∆238-245
FEATURES             Location/Qualifiers
     source          1..6413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..67
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        155..173
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    174..198
                     /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             213..235
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             242..898
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     856..878
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             908..931
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        1381..1399
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1400..1424
                     /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             1533..1577
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1615..1633)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1629..1676
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1713..2168
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1800..1819)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2010..2031
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2283..3074,3075..3143))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2906..2925
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3144..3236)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3317..3905
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3806..3825
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4059..4076
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4091..4230
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4156..4175)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4316..4338
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4332..4523)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5095..6177)
                     /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     6139..6158
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6178..6255)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
       61 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
      121 tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg
      181 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
      241 catgggctcc cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact
      301 tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga
      361 taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat
      421 tgatggtgat gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca
      481 caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt
      541 tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa
      601 agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca
      661 taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct
      721 tgatgttgtt ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt
      781 taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat
      841 agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaaca
      901 ggatccgctg gaagttctgt tccaggggcc cacagataac aacatccctc atggccagtg
      961 tgtcatctgc ctctatggtt tccaggagaa ggaggccttt accaaaacac cctgttacca
     1021 ctacttccac tgccactgcc ttgctcggta catccagcac atggagcaag agctgaaggc
     1081 acaaggacag gagcaggaac aggaacggca gcatgctaca accaaacaga aggcagtcgg
     1141 tgtgcagtgt ccagtgtgca gagagcccct cgtgtatgat cttgcctcac tgaaagcagc
     1201 ccctgaaccc caacagccca tggagctgta ccagcccagt gcagagagct tgcgccagca
     1261 agaagaacag gagcgggggg gaatcattga ccttgaggct gagcgataga agcttgcggc
     1321 cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta cacggccgca taatcgaaat
     1381 taatacgact cactataggg gaattgtgag cggataacaa ttccccatct tagtatatta
     1441 gttaagtata agaaggagat atacatatgg cagatctcaa ttggatatcg gccggccacg
     1501 cgatcgctga cgtcggtacc ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac
     1561 gccagcacat ggactcgtct actagcgcag cttaattaac ctaggctgct gccaccgctg
     1621 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1681 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1741 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1801 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1861 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1921 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1981 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2041 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2101 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     2161 tacaatttct ggcggcacga tggcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2221 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2281 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2341 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2401 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     2461 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2521 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2581 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     2641 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     2701 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     2761 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     2821 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     2881 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     2941 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3001 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3061 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3121 acacggaaat gttgaatact catactcttc ctttttcaat catgattgaa gcatttatca
     3181 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3241 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3301 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3361 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3421 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3481 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3541 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3601 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3661 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3721 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3781 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3841 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3901 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3961 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4021 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4081 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4141 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     4201 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     4261 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4321 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     4381 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     4441 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     4501 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4561 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4621 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4681 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4741 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4801 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4861 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     4921 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4981 ctagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5041 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     5101 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5161 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     5221 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     5281 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     5341 gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga
     5401 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     5461 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     5521 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     5581 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     5641 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     5701 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     5761 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     5821 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     5881 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     5941 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     6001 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     6061 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     6121 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     6181 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     6241 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     6301 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     6361 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccg
//
