LOCUS       sequence-399890-        5326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of AUP1 with GST fusion protein for binding 
            assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5326)
  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 5326)
  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 AUP1 292-410 5E
FEATURES             Location/Qualifiers
     source          1..5326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(169..174,175..190,191..197)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    205..221
                     /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             244..897
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     855..877
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             904..927
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1362..1384)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1529..1547)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1615..1719
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1720..1788,1789..2580)
                     /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(1938..1957)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2751..3339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3240..3259
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3493..3510
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3583..3660
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3661..4743
                     /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(3680..3699)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4792..4797,4798..4815,4816..4822)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4830..4846
                     /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     4854..4870
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4854..4870
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4866..5326,1..67)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4886..4903)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4886..4902)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4895..4917)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag
       61 gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt
      121 tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat
      181 catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca ggaaacagta
      241 ttcatgtccc ctatactagg ttattggaaa attaagggcc ttgtgcaacc cactcgactt
      301 cttttggaat atcttgaaga aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat
      361 aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc ttattatatt
      421 gatggtgatg ttaaattaac acagtctatg gccatcatac gttatatagc tgacaagcac
      481 aacatgttgg gtggttgtcc aaaagagcgt gcagagattt caatgcttga aggagcggtt
      541 ttggatatta gatacggtgt ttcgagaatt gcatatagta aagactttga aactctcaaa
      601 gttgattttc ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat
      661 aaaacatatt taaatggtga tcatgtaacc catcctgact tcatgttgta tgacgctctt
      721 gatgttgttt tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt
      781 aaaaaacgta ttgaagctat cccacaaatt gataagtact tgaaatccag caagtatata
      841 gcatggcctt tgcagggctg gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg
      901 gatctggaag ttctgttcca ggggcccctg ggatcctctc ctgatgtgca actggcaact
      961 ctggctcaga gagtcaagga agttttgccc catgtgccat tgggtgtcat ccagagagac
     1021 ctggccaaga ctggctgtgt agacttgact atcactaatc tgcttgaggg ggccgtagct
     1081 ttcatgcctg aagacatcac caagggaact cagtccctac ccacagcctc tgcctccaag
     1141 tttcccagct ctggcccggt gacccctcag ccaacagccc taacatttgc caagtcttcc
     1201 tgggccgagg aggagagcct gcaggagcgc gagcaagcac tatatgaata cgcagagagg
     1261 gagttcacag agagacgagc ccaggaggct gactgactcg agcggccgca tcgtgactga
     1321 ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     1381 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     1441 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     1501 ggagtgtata attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     1561 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1621 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1681 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     1741 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     1801 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     1861 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     1921 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     1981 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2041 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2101 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2161 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2221 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     2281 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     2341 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2401 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     2461 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2521 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     2581 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     2641 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     2701 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2761 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2821 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2881 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     2941 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3001 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3061 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3121 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3181 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3241 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3301 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3361 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3421 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3481 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3541 ctccttacgc atctgtgcgg tatttcacac cgcataaatt ccgacaccat cgaatggcgc
     3601 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     3661 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     3721 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     3781 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     3841 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     3901 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     3961 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     4021 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     4081 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     4141 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     4201 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     4261 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     4321 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     4381 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     4441 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     4501 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     4561 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     4621 gtctcactgg tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc ctctccccgc
     4681 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4741 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccccagg ctttacactt
     4801 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     4861 cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     4921 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     4981 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     5041 ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc
     5101 ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc
     5161 ccatctacac caacgtgacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga
     5221 atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc
     5281 agacgcgaat tatttttgat ggcgttggaa ttagcttatc gactgc
//
