LOCUS       sequence-399899-        5608 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RING domain-containing GST fusion protein for ubiquitination 
            assays..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5608)
  AUTHORS   Smith CE, Tsai YC, Liang YH, Khago D, Mariano J, Li J, Tarasov SG, 
            Gergel E, Tsai B, Villaneuva M, Clapp ME, Magidson V, Chari R, Byrd 
            RA, Ji X, Weissman AM
  TITLE     A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and
            is essential for ER-associated degradation.
  JOURNAL   PLoS Biol. 2021 Dec 8;19(12):e3001474. doi: 
            10.1371/journal.pbio.3001474. eCollection 2021 Dec.
  PUBMED    34879065
REFERENCE   2  (bases 1 to 5608)
  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"; journalName: "PLoS 
            Biol."; date: "2021-12-8"; volume: "19(12):e3001474. doi"; pages: " 
            10.1371/journal.pbio.3001474. eCollection 2021 Dec"
COMMENT     Sequence Label: pGEX-6P-1-HRD1 aa 236-444
FEATURES             Location/Qualifiers
     source          1..5608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(199..204,205..220,221..227)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    235..251
                     /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             274..927
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     885..907
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             934..957
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1674..1696)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1841..1859)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1927..2031
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2032..2100,2101..2892)
                     /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(2250..2269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3063..3651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3552..3571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3805..3822
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3895..3972
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3973..5055
                     /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(3992..4011)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5104..5109,5110..5127,5128..5134)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5142..5158
                     /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     5166..5182
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5166..5182
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5178..5608,1..97)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5198..5215)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5198..5214)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5207..5229)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tgatggcgtt ggaattagct tatcgactgc acggtgcacc aatgcttctg gcgtcaggca
       61 gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct
      121 caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa
      181 atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt ggaattgtga
      241 gcggataaca atttcacaca ggaaacagta ttcatgtccc ctatactagg ttattggaaa
      301 attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga aaaatatgaa
      361 gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt
      421 ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac acagtctatg
      481 gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt
      541 gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt ttcgagaatt
      601 gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct acctgaaatg
      661 ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga tcatgtaacc
      721 catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga cccaatgtgc
      781 ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat cccacaaatt
      841 gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg gcaagccacg
      901 tttggtggtg gcgaccatcc tccaaaatcg gatctggaag ttctgttcca ggggcccctg
      961 ggatccaagg tgcacacctt cccactcttt gccatccggc ccatgtacct ggccatgaga
     1021 cagttcaaga aagctgtgac agatgccatc atgtctcgcc gagccatccg caacatgaac
     1081 accctgtatc cagatgccac cccagaggag ctccaggcaa tggacaatgt ctgcatcatc
     1141 tgccgagaag agatggtgac tggtgccaag agactgccct gcaaccacat tttccatacc
     1201 agctgcctgc gctcctggtt ccagcggcag cagacctgcc ccacctgccg tatggatgtc
     1261 cttcgtgcat cgctgccagc gcagtcacca ccacccccgg agcctgcgga tcaggggcca
     1321 ccccctgccc cccacccccc accactcttg cctcagcccc ccaacttccc ccagggcctc
     1381 ctgcctcctt ttcctccagg catgttccca ctgtggcccc ccatgggccc ctttccacct
     1441 gtcccgcctc cccccagctc aggagaggct gtggctcctc catccaccag tgcagccctt
     1501 tctcggccca gtggagcagc tacaaccaca gctgctggca ccagtgctac tgctgcttct
     1561 gccacagcat ctggcccagg ctctggctct tgagaattcc cgggtcgact cgagcggccg
     1621 catcgtgact gactgacgat ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     1681 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     1741 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     1801 cgtagcgata gcggagtgta taattcttga agacgaaagg gcctcgtgat acgcctattt
     1861 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     1921 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1981 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     2041 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2101 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     2161 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     2221 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     2281 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2341 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2401 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2461 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2521 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     2581 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2641 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2701 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2761 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2821 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2881 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     2941 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3001 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3061 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3121 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3181 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3241 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3301 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3361 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3421 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3481 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3541 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3601 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3661 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3721 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3781 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     3841 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc
     3901 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     3961 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     4021 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     4081 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     4141 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     4201 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     4261 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     4321 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     4381 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     4441 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     4501 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     4561 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     4621 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     4681 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     4741 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     4801 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     4861 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     4921 cagctgttgc ccgtctcact ggtgaaaaga aaaaccacgc tagcgcccaa tacgcaaacc
     4981 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     5041 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacccca
     5101 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     5161 tcacacagga aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc
     5221 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     5281 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     5341 tgaatggcga atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc
     5401 tggagtgcga tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg
     5461 gttacgatgc gcccatctac accaacgtga cctatcccat tacggtcaat ccgccgtttg
     5521 ttcccacgga gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc
     5581 tacaggaagg ccagacgcga attatttt
//
