LOCUS       sequence-410481-        6008 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 6008)
  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 6008)
  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-gp78 RING
FEATURES             Location/Qualifiers
     source          1..6008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(185..190,191..206,207..213)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    221..237
                     /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             260..913
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     871..893
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             920..943
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2060..2082)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2227..2245)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2313..2417
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2418..2486,2487..3278)
                     /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(2636..2655)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3449..4037
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3938..3957
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4191..4208
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4281..4358
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4359..5441
                     /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(4378..4397)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5490..5495,5496..5513,5514..5520)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5528..5544
                     /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     5552..5568
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5552..5568
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5564..6008,1..83)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5584..5601)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5584..5600)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5593..5615)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttagcttatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg
       61 tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc
      121 gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga
      181 gctgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt
      241 cacacaggaa acagtattca tgtcccctat actaggttat tggaaaatta agggccttgt
      301 gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga
      361 gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa
      421 tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta
      481 tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat
      541 gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga
      601 ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga
      661 agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat
      721 gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc
      781 aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa
      841 atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga
      901 ccatcctcca aaatcggatc tggaagttct gttccagggg cccctgggat ccccaggaat
      961 tcgtcggcac aagaactatc tacgtgtggt tggaaacatg gaggccaggt ttgcagttgc
     1021 aactccagag gagctggctg tcaacaatga cgactgtgcc atctgttggg actccatgca
     1081 ggctgcgcgg aaactgccct gtggacatct tttccacaac tcctgtcttc gttcctggct
     1141 agaacaagac acctcctgtc caacatgcag aatgtctctt aatattgccg acaataatcg
     1201 tgtcagggaa gaatagcaag gagagaactt ggatgagaat ttggttcctg tagcagcagc
     1261 cgaagggaga cctcgcttaa accaacacaa tcacttcttc catttcgatg ggtctcggat
     1321 tgcgagctgg ctgccgagtt tttcggttga agtgatgcac accaccaaca ttcttggcat
     1381 tacgcaggcc agcaactccc agctcaatgc aatggctcat cagattcaag agatgtttcc
     1441 ccaggttcca taccatctgg tactgcagga cctccagctg acacgctcag ttgaaataac
     1501 aacagacaat attttagaag gacggattca agtacctttt cctacacagc ggtcagatag
     1561 catcagacct gcattgaaca gtcctgtgga aaggccaagc agtgaccagg aagagggaga
     1621 aacttctgct cagaccgagc gtgtgccact ggacctcagt cctcgcctgg aggagacgct
     1681 ggacttcggc gaggtggaag tggagcccag tgaggtggaa gacttcgagg ctcgtgggag
     1741 ccgcttctcc aagtctgctg atgagagaca gcgcatgctg gtgcagcgta aggacgaact
     1801 cctccagcaa gctcgcaaac gtttcttgaa caaaagttct gaagatgatg cggcctcaga
     1861 gagcttcctc ccctcggaag gtgcgtcctc tgaccccgtg accctgcgtc gaaggatgct
     1921 ggctgccgcc gcggaacgga ggcttcagaa gcagcagacc tcctagcgct cccttgcctt
     1981 cctcagctgg tcgactcgag cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt
     2041 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     2101 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     2161 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac
     2221 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     2281 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2341 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2401 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2461 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2521 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2581 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2641 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     2701 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2761 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2821 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2881 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2941 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3001 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3061 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3121 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3181 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3241 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3301 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3361 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3421 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3481 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3541 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     3601 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3661 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3721 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3781 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3841 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3901 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3961 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4021 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4081 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4141 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4201 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4261 tttcacaccg cataaattcc gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
     4321 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     4381 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     4441 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     4501 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     4561 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     4621 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     4681 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     4741 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     4801 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     4861 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     4921 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     4981 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     5041 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     5101 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     5161 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     5221 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     5281 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     5341 ccacgctagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5401 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
     5461 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5521 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga
     5581 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     5641 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     5701 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc
     5761 accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt
     5821 cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta
     5881 tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct
     5941 cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg
     6001 cgttggaa
//
