LOCUS       sequence-411031-        6028 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 6028)
  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 6028)
  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 61-410
FEATURES             Location/Qualifiers
     source          1..6028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     659..678
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(698..775)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(848..865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1019..1607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1099..1118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1778..2569,2570..2638))
                     /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     2401..2420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2639..2743)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2811..2829
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     2974..2996
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4133..4156)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(4163..4816)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(4183..4205)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     protein_bind    4839..4855
                     /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)."
     promoter        complement(join(4863..4869,4870..4885,4886..4891))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(4993..5520)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     5469..5491
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5483..5500
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5484..5500
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5516..5532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5516..5532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5540..5556
                     /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)."
     promoter        complement(join(5564..5570,5571..5588,5589..5594))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(join(5643..6028,1..697))
                     /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"
ORIGIN
        1 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
       61 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
      121 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
      181 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
      241 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
      301 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
      361 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
      421 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
      481 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
      541 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
      601 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
      661 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
      721 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtcggaat
      781 ttatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
      841 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
      901 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
      961 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1021 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     1081 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1141 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1201 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     1261 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1321 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1381 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1441 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1501 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     1561 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1621 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1681 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     1741 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     1801 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     1861 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     1921 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     1981 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2041 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca
     2101 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2161 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2221 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2281 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2341 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg
     2401 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2461 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     2521 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     2581 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     2641 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     2701 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     2761 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     2821 tcacgaggcc ctttcgtctt caagaattat acactccgct atcgctacgt gactgggtca
     2881 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2941 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3001 caccgtcatc accgaaacgc gcgaggcaga tcgtcagtca gtcacgatgc ggccgctcga
     3061 gtcagtcagc ctcctgggct cgtctctctg tgaatctcct tcttgcgtat tcatatagtg
     3121 cttgcttgcg ctcctgcagg ctctcctgcc gggcccagga agacttggca aatgttaggg
     3181 ctgttggctg aggggtcacc gggccagagc tgggaaactt ggaggcagag gctgtgggta
     3241 gggactgagt tcccttggtg atgtcttcag gcatgaaagc tacggccccc tcaagcagat
     3301 tagtgatagt caagtctaca cagccagtct tggccaggtc tctctggatg acacccaatg
     3361 gcacatgggg caaaacttcc ttgactctct gagccagagt tgccagttgc acatcaggag
     3421 aaggaccagg ggagggaggg aaagaagact gggctgactg ggggcgcaat ctggggtgtc
     3481 tttgtcgctt catgtgctct gctttgtcag ctggagtgag ccgtgtccct gtctggccca
     3541 attccttggc caccagctgt tgtacacgga gtgcaaactc ctcattcgct tcccctagtt
     3601 ggcgatgaac aggacgaagc caccttactt gatacaccgt gaaagggacg aaaagtgacc
     3661 acagcagttc tgagacccag gaggcatctg acaccgtcac agagaccagg ggtctctgaa
     3721 cttgcagggt aagaggttgt accacatctt ggatagaaaa tggccaggaa ctgaagcgca
     3781 ggagcccctc ccggccattg gtggcctctt cctcagggaa tagcagcaga ggagtggggg
     3841 gaagcctcgt ggaagcacag aatctcttga gtgactccac caactccccc cgcccattca
     3901 tctccatgaa gccccgagac cagcacacaa agctgggggg actattgagt agaggggtgc
     3961 tacaggtggt aagcaaattg actatgttgt ggtcgaaagg tgtcacatgg ttggaaatga
     4021 ggaccctgac actgtgatcc cggagtccgg agtcctcctg ccgggccacg agccctagca
     4081 ccgcacacat ggtccgcact acgaatctgc gaaggaattc cggggatccc aggggcccct
     4141 ggaacagaac ttccagatcc gattttggag gatggtcgcc accaccaaac gtggcttgcc
     4201 agccctgcaa aggccatgct atatacttgc tggatttcaa gtacttatca atttgtggga
     4261 tagcttcaat acgtttttta aaacaaacta attttgggaa cgcatccagg cacattgggt
     4321 ccatgtataa aacaacatca agagcgtcat acaacatgaa gtcaggatgg gttacatgat
     4381 caccatttaa atatgtttta tgacataaac gatcttcgaa cattttcagc atttcaggta
     4441 gcttgctaag aaaatcaact ttgagagttt caaagtcttt actatatgca attctcgaaa
     4501 caccgtatct aatatccaaa accgctcctt caagcattga aatctctgca cgctcttttg
     4561 gacaaccacc caacatgttg tgcttgtcag ctatataacg tatgatggcc atagactgtg
     4621 ttaatttaac atcaccatca atataataag gaagattggg aaactccaaa cccaattcaa
     4681 actttttgtt tcgccattta tcaccttcat cgcgctcata caaatgctct tcatattttt
     4741 cttcaagata ttccaaaaga agtcgagtgg gttgcacaag gcccttaatt ttccaataac
     4801 ctagtatagg ggacatgaat actgtttcct gtgtgaaatt gttatccgct cacaattcca
     4861 cacattatac gagccgatga ttaattgtca acagctcatt tcagaatatt tgccagaacc
     4921 gttatgatgt cggcgcaaaa aacattatcc agaacgggag tgcgccttga gcgacacgaa
     4981 ttatgcagtg atttacgacc tgcacagcca taccacagct tccgatggct gcctgacgcc
     5041 agaagcattg gtgcaccgtg cagtcgataa gctaattcca acgccatcaa aaataattcg
     5101 cgtctggcct tcctgtagcc agctttcatc aacattaaat gtgagcgagt aacaacccgt
     5161 cggattctcc gtgggaacaa acggcggatt gaccgtaatg ggataggtca cgttggtgta
     5221 gatgggcgca tcgtaaccgt gcatctgcca gtttgagggg acgacgacag tatcggcctc
     5281 aggaagatcg cactccagcc agctttccgg caccgcttct ggtgccggaa accaggcaaa
     5341 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     5401 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     5461 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaatccgtaa tcatggtcat
     5521 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5581 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta acttacatta attgcgttgc
     5641 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5701 aacgcgcggg gagaggcggt ttgcgtattg ggcgctagcg tggtttttct tttcaccagt
     5761 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5821 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5881 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5941 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     6001 atcgcagtgg gaacgatgcc ctcattca
//
