LOCUS       sequence-275582-        6437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GST tagged AO7 encoding aa 126-258 with an I137A mutation for 
            expression in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6437)
  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 6437)
  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 I137A
FEATURES             Location/Qualifiers
     source          1..6437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     90..109
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        197..215
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    216..240
                     /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             255..277
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             284..940
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     898..920
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             950..973
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        1447..1465
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1466..1490
                     /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             1599..1643
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1681..1699)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1695..1742
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1779..2234
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1866..1885)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2076..2097
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2349..3140,3141..3209))
                     /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     2972..2991
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3210..3302)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3383..3971
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3872..3891
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4125..4142
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4157..4296
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4222..4241)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4382..4404
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4398..4589)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5161..6243)
                     /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     6205..6224
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6244..6321)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
       61 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
      121 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat
      181 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
      241 cctctagaaa taattttgtt taactttaag aaggagatat accatgggct cccctatact
      301 aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg aatatcttga
      361 agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa
      421 gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg atgttaaatt
      481 aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt tgggtggttg
      541 tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata ttagatacgg
      601 tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt ttcttagcaa
      661 gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat atttaaatgg
      721 tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg ttttatacat
      781 ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac gtattgaagc
      841 tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc ctttgcaggg
      901 ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa caggatccgc tggaagttct
      961 gttccagggg cccacagata acaacatccc tcatggccag tgtgtcgcct gcctctatgg
     1021 tttccaggag aaggaggcct ttaccaaaac accctgttac cactacttcc actgccactg
     1081 ccttgctcgg tacatccagc acatggagca agagctgaag gcacaaggac aggagcagga
     1141 acaggaacgg cagcatgcta caaccaaaca gaaggcagtc ggtgtgcagt gtccagtgtg
     1201 cagagagccc ctcgtgtatg atcttgcctc actgaaagca gcccctgaac cccaacagcc
     1261 catggagctg taccagccca gtgcagagag cttgcgccag caagaagaac gcaagcggct
     1321 ctaccagagg cagcaggagc gggggggaat cattgacctt gaggctgagc gatagaagct
     1381 tgcggccgca taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat
     1441 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt
     1501 atattagtta agtataagaa ggagatatac atatggcaga tctcaattgg atatcggccg
     1561 gccacgcgat cgctgacgtc ggtaccctcg agtctggtaa agaaaccgct gctgcgaaat
     1621 ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag gctgctgcca
     1681 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1741 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1801 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1861 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1921 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1981 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2041 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2101 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2161 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2221 aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat cttcacctag
     2281 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2341 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2401 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2461 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2521 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2581 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2641 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     2701 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     2761 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2821 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     2881 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     2941 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3001 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3061 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3121 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3181 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg attgaagcat
     3241 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3301 aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3361 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3421 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3481 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3541 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3601 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3661 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3721 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3781 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3841 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3901 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3961 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4021 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4081 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4141 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4201 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4261 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4321 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4381 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4441 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4501 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4561 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4621 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4681 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4741 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4801 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4861 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4921 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4981 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     5041 atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5101 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     5161 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5221 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5281 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     5341 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     5401 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     5461 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     5521 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     5581 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     5641 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     5701 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     5761 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5821 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     5881 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     5941 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6001 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     6061 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     6121 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     6181 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     6241 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     6301 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     6361 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     6421 gcaaggagat ggcgccc
//
