LOCUS       sequence-384513-        6599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for co-expression of the AUP1 G2BR with UBE2G2 for 
            crystallography.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6599)
  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 6599)
  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: pETDUET-GST-AUP1 379-410:Ube2G2
FEATURES             Location/Qualifiers
     source          1..6599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     199..218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     452..469
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    484..623
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(549..568)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     709..731
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(725..916)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1488..2570)
                     /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     2532..2551
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2571..2648)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2854..2873
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2961..2979
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2980..3004
                     /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)."
     RBS             3019..3041
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3048..3704
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     3662..3684
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             3714..3737
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        3929..3947
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3948..3972
                     /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             4525..4569
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4607..4625)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4621..4668
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4705..5160
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4792..4811)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5002..5023
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5275..6066,6067..6135))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5898..5917
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6136..6228)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6309..6599,1..298)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
       61 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
      121 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
      181 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      241 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      301 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      361 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      421 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      481 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
      541 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
      601 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
      661 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
      721 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
      781 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
      841 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
      901 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
      961 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     1021 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     1081 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     1141 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     1201 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     1261 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     1321 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgctagtca
     1381 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     1441 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     1501 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     1561 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     1621 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     1681 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     1741 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta
     1801 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     1861 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     1921 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     1981 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     2041 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     2101 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     2161 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     2221 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     2281 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     2341 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     2401 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     2461 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     2521 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     2581 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     2641 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     2701 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     2761 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     2821 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     2881 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     2941 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     3001 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggctccccta
     3061 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
     3121 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
     3181 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
     3241 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
     3301 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
     3361 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
     3421 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
     3481 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
     3541 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
     3601 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
     3661 agggctggca agccacgttt ggtggtggcg accatcctcc aaaacaggat ccgctggaag
     3721 ttctgttcca ggggcccgcc aagtcttcct gggcccggca ggagagcctg caggagcgca
     3781 agcaagcact atatgaatac gcaagaagga gattcacaga gagacgagcc caggaggctg
     3841 actgaaggct tctcgacaag cttgcggccg cataatgctt aagtcgaaca gaaagtaatc
     3901 gtattgtaca cggccgcata atcgaaatta atacgactca ctatagggga attgtgagcg
     3961 gataacaatt ccccatctta gtatattagt taagtataag aaggagatat acatatggcg
     4021 gggacggcgt tgaagaggct gatggccgag tataagcaat taaccctgaa tcctccagaa
     4081 ggaatcgtgg caggccccat gaatgaagag aatttttttg aatgggaggc attgatcatg
     4141 ggccctgaag acacttgttt tgagtttggt gtttttcccg ccatcctgag tttcccactt
     4201 gactacccct tgagccctcc gaagatgaga ttcacctgtg agatgttcca tcccaacatc
     4261 tatcctgatg gcagagtctg catctccatc ctgcatgctc caggtgatga tcccatgggt
     4321 tatgagagca gtgccgagcg gtggagccca gtgcagagtg tggagaagat cctgctttct
     4381 gttgtgagca tgctggcaga gcccaacgat gagagtggag caaacgtaga tgcttccaag
     4441 atgtggcggg atgaccggga gcagttctac aagatcgcca agcagattgt ccagaagtct
     4501 ctggggctct gactcgagtc tggtaaagaa accgctgctg cgaaatttga acgccagcac
     4561 atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc tgagcaataa
     4621 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     4681 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4741 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4801 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4861 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4921 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4981 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5041 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5101 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     5161 ctggcggcac gatggcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5221 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5281 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5341 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5401 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5461 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5521 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5581 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5641 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5701 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5761 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5821 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5881 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5941 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6001 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6061 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6121 atgttgaata ctcatactct tcctttttca atcatgattg aagcatttat cagggttatt
     6181 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggtcatgacc
     6241 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6301 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6361 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6421 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     6481 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6541 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtt
//
