LOCUS       sequence-273792-        5749 bp    DNA     circular SYN 14-APR-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 5749)
  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 5749)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pGEX6p1 AO7RE
FEATURES             Location/Qualifiers
     source          1..5749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1617..1639)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1784..1802)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1870..1974
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1975..2043,2044..2835)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2193..2212)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3006..3594
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3495..3514
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3748..3765
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3838..3915
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3916..4998
                     /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     complement(3935..3954)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5047..5052,5053..5070,5071..5077)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5085..5101
                     /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)."
     primer_bind     5109..5125
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5109..5125
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5121..5648
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5141..5158)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5141..5157)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5150..5172)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggatcccc ggaattcgcg
      781 gcgtctgcgt ctgcagctgc aggggaggag gactgggtcc ttccctctga agttgaagta
      841 ttggagtcca tctatctaga tgaactacag gtgattaaag gaaatggcag aacttcacca
      901 tgggagatct acatcacttt gcatcctgcc actgcagagg accaggattc acagtatgtc
      961 tgcttcactc tggtgcttca ggtcccagca gagtatcccc atgaggtgcc acagatctct
     1021 atccgaaatc cccgaggact ttcagatgaa cagatccaca cgatcttaca ggtgctgggc
     1081 cacgtggcca aggctgggct gggcactgcc atgctgtatg aactcattga gaaagggaag
     1141 gaaattctca cagataacaa catccctcat ggccagtgtg tcatctgcct ctatggtttc
     1201 caggagaagg aggcctttac caaaacaccc tgttaccact acttccactg ccactgcctt
     1261 gctcggtaca tccagcacat ggagcaagag ctgaaggcac aaggacagga gcaggaacag
     1321 gaacggcagc atgctacaac caaacagaag gcagtcggtg tgcagtgtcc agtgtgcaga
     1381 gagcccctcg tgtatgatct tgcctcactg aaagcagccc ctgaacccca acagcccatg
     1441 gagctgtacc agcccagtgc agagagcttg cgccagcaag aagaacgcaa gcggctctac
     1501 cagaggcagc aggagcgggg gggaatcatt gaccttgagg ctgagcgata gctcgagcgg
     1561 ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     1621 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     1681 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     1741 tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta
     1801 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     1861 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1921 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     1981 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2041 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2101 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2161 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     2221 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2281 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2341 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2401 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2461 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     2521 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2581 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2641 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2701 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2761 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2821 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     2881 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2941 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3001 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3061 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3121 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3181 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3241 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3301 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3361 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3421 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3481 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3541 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3601 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3661 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3721 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3781 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac
     3841 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     3901 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     3961 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     4021 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     4081 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     4141 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     4201 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     4261 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     4321 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     4381 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     4441 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     4501 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     4561 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     4621 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     4681 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
     4741 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     4801 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     4861 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca cgctagcgcc caatacgcaa
     4921 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4981 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     5041 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     5101 atttcacaca ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac
     5161 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     5221 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     5281 gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct
     5341 ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc
     5401 acggttacga tgcgcccatc tacaccaacg tgacctatcc cattacggtc aatccgccgt
     5461 ttgttcccac ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct
     5521 ggctacagga aggccagacg cgaattattt ttgatggcgt tggaattagc ttatcgactg
     5581 cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca
     5641 ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt
     5701 ttttgcgccg acatcataac ggttctggca aatattctga aatgagctg
//
