LOCUS       sequence-275605-        6437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GST tagged AO7 encoding aa 126-258 with a Y165A 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 Y165A
FEATURES             Location/Qualifiers
     source          1..6437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..24
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        112..130
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    131..155
                     /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             170..192
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             199..855
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     813..835
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             865..888
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        1362..1380
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1381..1405
                     /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             1514..1558
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1596..1614)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1610..1657
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1694..2149
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1781..1800)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1991..2012
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2264..3055,3056..3124))
                     /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     2887..2906
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3125..3217)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3298..3886
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3787..3806
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4040..4057
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4072..4211
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4137..4156)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4297..4319
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4313..4504)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5076..6158)
                     /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     6120..6139
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6159..6236)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
       61 ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac
      121 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
      181 ttaagaagga gatataccat gggctcccct atactaggtt attggaaaat taagggcctt
      241 gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat
      301 gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc
      361 aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt
      421 tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca
      481 atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa
      541 gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc
      601 gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc
      661 atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc
      721 ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg
      781 aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc
      841 gaccatcctc caaaacagga tccgctggaa gttctgttcc aggggcccac agataacaac
      901 atccctcatg gccagtgtgt catctgcctc tatggtttcc aggagaagga ggcctttacc
      961 aaaacaccct gttaccacta cttccactgc cactgccttg ctcgggccat ccagcacatg
     1021 gagcaagagc tgaaggcaca aggacaggag caggaacagg aacggcagca tgctacaacc
     1081 aaacagaagg cagtcggtgt gcagtgtcca gtgtgcagag agcccctcgt gtatgatctt
     1141 gcctcactga aagcagcccc tgaaccccaa cagcccatgg agctgtacca gcccagtgca
     1201 gagagcttgc gccagcaaga agaacgcaag cggctctacc agaggcagca ggagcggggg
     1261 ggaatcattg accttgaggc tgagcgatag aagcttgcgg ccgcataatg cttaagtcga
     1321 acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg
     1381 ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga
     1441 tatacatatg gcagatctca attggatatc ggccggccac gcgatcgctg acgtcggtac
     1501 cctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc
     1561 tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc
     1621 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1681 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1741 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1801 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1861 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1921 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1981 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2041 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2101 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc tggcggcacg
     2161 atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2221 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2281 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     2341 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     2401 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     2461 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     2521 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     2581 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     2641 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     2701 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     2761 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     2821 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     2881 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     2941 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3001 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3061 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3121 tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg tctcatgagc
     3181 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca aaatccctta
     3241 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3301 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3361 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3421 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3481 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3541 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3601 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3661 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3721 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3781 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3841 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3901 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3961 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4021 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4081 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     4141 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4201 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4261 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4321 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4381 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4441 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     4501 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     4561 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     4621 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4681 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4741 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4801 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4861 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4921 gccagcctag ccgggtcctc aacgacagga gcacgatcat gctagtcatg ccccgcgccc
     4981 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5041 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5101 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5161 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5221 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5281 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5341 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     5401 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5461 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5521 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5581 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5641 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5701 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5761 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5821 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5881 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5941 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     6001 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     6061 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     6121 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     6181 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     6241 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     6301 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     6361 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     6421 gcgagcccga tcttccc
//
