LOCUS       sequence-399883-        5335 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 5335)
  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 5335)
  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 A397D
FEATURES             Location/Qualifiers
     source          1..5335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(524..529,530..547,548..554)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    562..578
                     /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     586..602
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     586..602
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             598..1125
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(618..635)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(618..634)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(627..649)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1227..1232,1233..1248,1249..1255)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1263..1279
                     /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             1302..1955
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1913..1935
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1962..1985
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2429..2451)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2596..2614)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2682..2786
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2787..2855,2856..3647)
                     /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(3005..3024)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3818..4406
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4307..4326
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4560..4577
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4650..4727
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4728..5335,1..475)
                     /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(4747..4766)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
       61 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
      121 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
      181 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
      241 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
      301 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
      361 ggtgaaaaga aaaaccacgc tagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      481 acgcaattaa tgtaagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      541 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      601 accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
      661 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
      721 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt
      781 gcctggtttc cggcaccaga agcggtgccg gaaagctggc tggagtgcga tcttcctgag
      841 gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc gcccatctac
      901 accaacgtga cctatcccat tacggtcaat ccgccgtttg ttcccacgga gaatccgacg
      961 ggttgttact cgctcacatt taatgttgat gaaagctggc tacaggaagg ccagacgcga
     1021 attatttttg atggcgttgg aattagctta tcgactgcac ggtgcaccaa tgcttctggc
     1081 gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc
     1141 gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca tcataacggt
     1201 tctggcaaat attctgaaat gagctgttga caattaatca tcggctcgta taatgtgtgg
     1261 aattgtgagc ggataacaat ttcacacagg aaacagtatt catgtcccct atactaggtt
     1321 attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat cttgaagaaa
     1381 aatatgaaga gcatttgtat gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg
     1441 aattgggttt ggagtttccc aatcttcctt attatattga tggtgatgtt aaattaacac
     1501 agtctatggc catcatacgt tatatagctg acaagcacaa catgttgggt ggttgtccaa
     1561 aagagcgtgc agagatttca atgcttgaag gagcggtttt ggatattaga tacggtgttt
     1621 cgagaattgc atatagtaaa gactttgaaa ctctcaaagt tgattttctt agcaagctac
     1681 ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa aacatattta aatggtgatc
     1741 atgtaaccca tcctgacttc atgttgtatg acgctcttga tgttgtttta tacatggacc
     1801 caatgtgcct ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt gaagctatcc
     1861 cacaaattga taagtacttg aaatccagca agtatatagc atggcctttg cagggctggc
     1921 aagccacgtt tggtggtggc gaccatcctc caaaatcgga tctggaagtt ctgttccagg
     1981 ggcccctggg atccccggaa ttctctcctg atgtgcaact ggcaactctg gctcagagag
     2041 tcaaggaagt tttgccccat gtgccattgg gtgtcatcca gagagacctg gccaagactg
     2101 gctgtgtaga cttgactatc actaatctgc ttgagggggc cgtagctttc atgcctgaag
     2161 acatcaccaa gggaactcag tccctaccca cagcctctgc ctccaagttt cccagctctg
     2221 gcccggtgac ccctcagcca acagccctaa catttgccaa gtcttcctgg gcccggcagg
     2281 agagcctgca ggagcgcaag caagcactat atgaatacga cagaaggaga ttcacagaga
     2341 gacgagccca ggaggctgac tgactcgagc ggccgcatcg tgactgactg acgatctgcc
     2401 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     2461 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     2521 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtataatt
     2581 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     2641 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2701 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2761 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2821 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2881 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2941 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3001 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     3061 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3121 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3181 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3241 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3301 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     3361 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3421 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3481 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3541 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3601 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3661 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3721 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3781 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3841 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3901 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3961 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4021 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4081 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4141 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4201 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4261 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4321 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4381 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4441 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4501 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4561 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4621 tgtgcggtat ttcacaccgc ataaattccg acaccatcga atggcgcaaa acctttcgcg
     4681 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     4741 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     4801 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     4861 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     4921 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     4981 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     5041 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     5101 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     5161 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     5221 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     5281 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataa
//
