LOCUS       sequence-383992-        6611 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Assessing domain requirements of AUP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6611)
  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 6611)
  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-DCUE
FEATURES             Location/Qualifiers
     source          1..6611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..48)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        220..599
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        600..803
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     754..774
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     848..867
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        848..866
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2036..2059
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2069..2086
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2149..2167)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2150..2167)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2187..2204)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2193..2417
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2463..2891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2550..2569)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2760..2781
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2900..2920)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2905..3234
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3085..3220
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3147..3166
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3301..4095
                     /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(3355..3374)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3965..3984
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4269..4390
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4306..4325)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4360..4379
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4439..4455)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4439..4455)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4452..4474)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4463..4479
                     /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(4487..4493,4494..4511,4512..4517))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4532..4553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4670..4687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4841..5429)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4921..4940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5600..6391,6392..6460))
                     /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     6223..6242
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6461..6565)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
       61 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
      121 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
      181 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
      241 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      301 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      361 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      421 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      481 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      541 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      601 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
      661 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      721 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      781 gggaggtcta tataagcaga gctctctggc taactagaga acccactgct tactggctta
      841 tcgaaattaa tacgactcac tatagggaga cccaagcttg gtaccgagct cggatccact
      901 agtaacggcc gccagtgtgc tggaattcat ggagcttccc tcagggccgg ggccggagcg
      961 gctctttgac tcgcaccggc ttccgggtga ctgcttccta ctgctcgtgc tgctgctcta
     1021 cgcgccagtc gggttctgcc tcctcgtcct gcgcctcttt ctcgggatcc acgtcttcct
     1081 ggtcagctgc gcgctgccag acagcgtcct tcgcagattc gtagtgcgga ccatgtgtgc
     1141 ggtgctaggg ctcgtggccc ggcaggagga ctccggactc cgggatcaca gtgtcagggt
     1201 cctcatttcc aaccatgtga cacctttcga ccacaacata gtcaatttgc ttaccacctg
     1261 tagcacccct ctactcaata gtccccccag ctttgtgtgc tggtctcggg gcttcatgga
     1321 gatgaatggg cggggggagt tggtggagtc actcaagaga ttctgtgctt ccacgaggct
     1381 tccccccact cctctgctgc tattccctga ggaagaggcc accaatggcc gggaggggct
     1441 cctgcgcttc agttcctggc cattttctat ccaagatgtg gtacaacctc ttaccctgca
     1501 agttcagaga cccctggtct ctgtgacggt gtcagatgcc tcctgggtct cagaactgct
     1561 gtggtcactt ttcgtccctt tcacggtgta tcaagtaagg tggcttcgtc ctgttcatcg
     1621 ccaactaggg gaagcgaatg aggagtttgc actccgtgta caacagctgg tggccaagga
     1681 attgggccag acagggacac ggctcactcc agctgacaaa gcagagcaca tgaagcgaca
     1741 aagacacccc agattgcgcc cccagtcagc ccagtcttct ttccctccct cccctggtcc
     1801 ttctgtagct ttcatgcctg aagacatcac caagggaact cagtccctac ccacagcctc
     1861 tgcctccaag tttcccagct ctggcccggt gacccctcag ccaacagccc taacatttgc
     1921 caagtcttcc tgggcccggc aggagagcct gcaggagcgc aagcaagcac tatatgaata
     1981 cgcaagaagg agattcacag agagacgagc ccaggaggct gacctcgagg tcgaggacta
     2041 caaggacgac gatgacaaga tcttcgagca ccaccaccac caccactgag atccggctgc
     2101 taacaaagcc cgaaaggaag ctgagttggc tgctctagag ggccctattc tatagtgtca
     2161 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2221 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2281 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2341 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2401 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2461 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2521 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2581 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2641 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2701 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2761 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2821 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2881 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2941 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3001 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3061 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3121 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3181 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3241 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     3301 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3361 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3421 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3481 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3541 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3601 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3661 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3721 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3781 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3841 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3901 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3961 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4021 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4081 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4141 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4201 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4261 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4321 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4381 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4441 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4501 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4561 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4621 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4681 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4741 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4801 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4861 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4921 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4981 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5041 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5101 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5161 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5221 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5281 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5341 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5401 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5461 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5521 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5581 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5641 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5701 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5761 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5821 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5881 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5941 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6001 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6061 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6121 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6181 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6241 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6301 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6361 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6421 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6481 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6541 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     6601 gatcgggaga t
//
