LOCUS       sequence-422719-        6437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GST tagged AO7 encoding aa 126-258 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 AO7REwt
FEATURES             Location/Qualifiers
     source          1..6437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             71..727
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     685..707
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             737..760
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        1234..1252
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1253..1277
                     /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             1386..1430
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1468..1486)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1482..1529
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1566..2021
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1653..1672)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1863..1884
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2136..2927,2928..2996))
                     /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     2759..2778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2997..3089)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3170..3758
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3659..3678
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3912..3929
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3944..4083
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4009..4028)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4169..4191
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4185..4376)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4948..6030)
                     /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     5992..6011
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6031..6108)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6314..6333
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6421..6437,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggctccc ctatactagg ttattggaaa attaagggcc ttgtgcaacc
      121 cactcgactt cttttggaat atcttgaaga aaaatatgaa gagcatttgt atgagcgcga
      181 tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc
      241 ttattatatt gatggtgatg ttaaattaac acagtctatg gccatcatac gttatatagc
      301 tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt gcagagattt caatgcttga
      361 aggagcggtt ttggatatta gatacggtgt ttcgagaatt gcatatagta aagactttga
      421 aactctcaaa gttgattttc ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg
      481 tttatgtcat aaaacatatt taaatggtga tcatgtaacc catcctgact tcatgttgta
      541 tgacgctctt gatgttgttt tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt
      601 agtttgtttt aaaaaacgta ttgaagctat cccacaaatt gataagtact tgaaatccag
      661 caagtatata gcatggcctt tgcagggctg gcaagccacg tttggtggtg gcgaccatcc
      721 tccaaaacag gatccgctgg aagttctgtt ccaggggccc acagataaca acatccctca
      781 tggccagtgt gtcatctgcc tctatggttt ccaggagaag gaggccttta ccaaaacacc
      841 ctgttaccac tacttccact gccactgcct tgctcggtac atccagcaca tggagcaaga
      901 gctgaaggca caaggacagg agcaggaaca ggaacggcag catgctacaa ccaaacagaa
      961 ggcagtcggt gtgcagtgtc cagtgtgcag agagcccctc gtgtatgatc ttgcctcact
     1021 gaaagcagcc cctgaacccc aacagcccat ggagctgtac cagcccagtg cagagagctt
     1081 gcgccagcaa gaagaacgca agcggctcta ccagaggcag caggagcggg ggggaatcat
     1141 tgaccttgag gctgagcgat agaagcttgc ggccgcataa tgcttaagtc gaacagaaag
     1201 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
     1261 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
     1321 tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt
     1381 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
     1441 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1501 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1561 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1621 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1681 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1741 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1801 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1861 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1921 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1981 atttaacgcg aattttaaca aaatattaac gtttacaatt tctggcggca cgatggcatg
     2041 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2101 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2161 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2221 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2281 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2341 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2401 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     2461 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2521 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     2581 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     2641 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     2701 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     2761 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     2821 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     2881 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     2941 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3001 ttcctttttc aatcatgatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3061 atttgaatgt atttagaaaa ataaacaaat aggtcatgac caaaatccct taacgtgagt
     3121 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3181 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3241 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3301 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3361 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3421 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3481 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3541 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3601 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3661 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3721 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3781 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3841 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3901 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3961 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     4021 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     4081 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4141 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4201 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     4261 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     4321 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     4381 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     4441 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     4501 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     4561 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     4621 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     4681 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     4741 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     4801 agccgggtcc tcaacgacag gagcacgatc atgctagtca tgccccgcgc ccaccggaag
     4861 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4921 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4981 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5041 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     5101 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     5161 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     5221 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5281 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5341 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5401 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5461 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5521 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5581 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5641 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5701 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5761 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     5821 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5881 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5941 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6001 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     6061 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     6121 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     6181 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     6241 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     6301 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     6361 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat
     6421 taatacgact cactata
//
