LOCUS       sequence-428773-        6011 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Assessing domain requirement sof AUP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6011)
  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 6011)
  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: pCDNA3-AUP1-FLAG-DATDCUE
FEATURES             Location/Qualifiers
     source          1..6011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(300..1091,1092..1160))
                     /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     923..942
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1161..1265)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1340..1359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1531..1910
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1911..2114
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2065..2085
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2159..2178
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2159..2177
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2747..2770
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2780..2797
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2860..2878)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2861..2878)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2898..2915)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2904..3128
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3174..3602
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3261..3280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3471..3492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3616..3945
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3796..3931
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3858..3877
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4012..4806
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4066..4085)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4676..4695
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4980..5101
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5017..5036)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5071..5090
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5150..5166)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5150..5166)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5163..5185)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5174..5190
                     /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(5198..5204,5205..5222,5223..5228))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5243..5264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5381..5398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5552..6011,1..129))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5632..5651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
       61 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
      121 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
      181 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
      241 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
      301 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
      361 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
      421 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
      481 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
      541 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
      601 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
      661 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
      721 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
      781 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
      841 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
      901 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
      961 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     1021 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     1081 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     1141 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     1201 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     1261 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     1321 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     1381 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     1441 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     1501 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     1561 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     1621 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     1681 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     1741 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     1801 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     1861 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     1921 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     1981 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     2041 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     2101 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
     2161 atacgactca ctatagggag acccaagctt ggtaccgagc tcggatccac tagtaacggc
     2221 cgccagtgtg ctggaattca tggagcttcc ctcagggccg gggccggagc ggctctttga
     2281 ctcgcaccgt cttccgggtg actgcttcct actgctcgtg ctgctgctct acgcgccagt
     2341 cgggttctgc ctcctcgtcc tgcgcctctt tctcgggatc cacgtcttcc tggtcagctg
     2401 cgcgctgcca gacagcgtcc ttcgcagatt cgtagtgcgg accatgtgtg cggtgctagg
     2461 gctcgtggcc cggcaggagg actccggact ccgggatcac agtgtcagag aattcgtagc
     2521 tttcatgcct gaagacatca ccaagggaac tcagtcccta cccacagcct ctgcctccaa
     2581 gtttcccagc tctggcccgg tgacccctaa gccaacagcc ctaacatttg ccaagtcttc
     2641 ctgggcccgg caggagagcc tgcaggagcg caagcaagca ctatatgaat acgcaagaag
     2701 gagattcaca gagagacgag cccaggaggc tgacctcgag gtcgaggact acaaggacga
     2761 cgatgacaag atcttcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
     2821 ccgaaaggaa gctgagttgg ctgctctaga gggccctatt ctatagtgtc acctaaatgc
     2881 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2941 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3001 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3061 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3121 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3181 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3241 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3301 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3361 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3421 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3481 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3541 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3601 ttaattctgt ggaatgtgtg tcagttatgg tgtggaaagt ccccaggctc cccagcaggc
     3661 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3721 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3781 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3841 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3901 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3961 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     4021 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4081 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4141 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4201 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4261 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4321 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4381 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4441 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4501 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4561 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4621 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4681 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4741 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4801 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4861 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4921 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4981 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5041 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5101 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5161 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5221 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5281 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5341 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5401 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5461 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5521 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5581 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5641 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5701 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5761 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5821 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5881 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5941 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6001 ttggtatctg c
//
