LOCUS       sequence-399902-        5428 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 5428)
  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 5428)
  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-4T-3-TRC8 513-664
FEATURES             Location/Qualifiers
     source          1..5428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(192..197,198..213,214..220)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    228..244
                     /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             267..920
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     878..900
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             927..944
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1487..1509)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1654..1672)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1740..1844
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1845..1913,1914..2705)
                     /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(2063..2082)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2876..3464
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3365..3384
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3618..3635
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3708..3785
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3786..4868
                     /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(3805..3824)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4917..4922,4923..4940,4941..4947)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4955..4971
                     /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     4979..4995
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4979..4995
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4991..5428,1..90)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5011..5028)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5011..5027)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5020..5042)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gttggaatta gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg
       61 gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg
      121 cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct
      181 gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata
      241 acaatttcac acaggaaaca gtattcatgt cccctatact aggttattgg aaaattaagg
      301 gccttgtgca acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt
      361 tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt
      421 ttcccaatct tccttattat attgatggtg atgttaaatt aacacagtct atggccatca
      481 tacgttatat agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga
      541 tttcaatgct tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata
      601 gtaaagactt tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa
      661 tgttcgaaga tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg
      721 acttcatgtt gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg
      781 cgttcccaaa attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt
      841 acttgaaatc cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg
      901 gtggcgacca tcctccaaaa tcggatctgg ttccgcgtgg atccaccaaa aatggctgga
      961 agacatttat gaatcgtagg actgctgtga ataaaattaa ttcacttcct gaaataaaag
     1021 ggagccgctt acaagaaata aatgatgtat gtgcaatctg ctatcatgag tttacaacat
     1081 ctgctcgtat tacaccgtgt aatcattatt tccatgcact ttgccttcgg aaatggctgt
     1141 acattcaaga tacttgtcca atgtgccatc agaaagtata catcgaagat gatatcaagg
     1201 ataattcaaa tgtatctaac aacaatggat ttattccacc caatgaaact ccagaggaag
     1261 ctgtaagaga agctgctgct gaatctgaca gggaattgaa cgaagatgac agtacagatt
     1321 gtgatgatga tgttcaaaga gaaagaaatg gagtgattca gcacacaggc gcagcagctg
     1381 aagaatttaa tgatgatact gactgagaat tcccgggtcg actcgagcgg ccgcatcgtg
     1441 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     1501 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     1561 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     1621 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     1681 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     1741 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1801 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1861 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1921 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1981 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2041 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     2101 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2161 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2221 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2281 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2341 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     2401 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2461 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2521 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2581 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2641 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     2701 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     2761 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2821 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2881 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2941 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3001 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3061 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3121 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3181 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3241 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3301 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3361 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3421 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3481 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3541 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3601 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3661 attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat
     3721 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     3781 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     3841 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     3901 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     3961 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     4021 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     4081 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     4141 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     4201 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     4261 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     4321 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     4381 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     4441 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     4501 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     4561 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     4621 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     4681 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     4741 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     4801 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     4861 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta
     4921 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     4981 ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg
     5041 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     5101 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     5161 cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg
     5221 cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga
     5281 tgcgcccatc tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac
     5341 ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga
     5401 aggccagacg cgaattattt ttgatggc
//
