LOCUS       sequence-382171-        6746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of AUP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6746)
  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 6746)
  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
FEATURES             Location/Qualifiers
     source          1..6746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        191..570
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        571..774
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     725..745
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     819..838
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        819..837
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2142..2165
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2175..2192
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2255..2273)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2256..2273)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2293..2310)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2299..2523
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2569..2997
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2656..2675)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2866..2887
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3006..3026)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3011..3340
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3191..3326
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3253..3272
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3407..4201
                     /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(3461..3480)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4071..4090
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4375..4496
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4412..4431)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4466..4485
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4545..4561)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4545..4561)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4558..4580)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4569..4585
                     /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(4593..4599,4600..4617,4618..4623))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4638..4659
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4776..4793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4947..5535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5027..5046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5706..6497,6498..6566))
                     /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     6329..6348
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6567..6671)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(6746,1..19))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
       61 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
      121 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
      181 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      241 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      301 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      361 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      421 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      481 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      541 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      601 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      661 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      721 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
      781 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
      841 acccaagctt ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattca
      901 tggagcttcc ctcagggccg gggccggagc ggctctttga ctcgcaccgg cttccgggtg
      961 actgcttcct actgctcgtg ctgctgctct acgcgccagt cgggttctgc ctcctcgtcc
     1021 tgcgcctctt tctcgggatc cacgtcttcc tggtcagctg cgcgctgcca gacagcgtcc
     1081 ttcgcagatt cgtagtgcgg accatgtgtg cggtgctagg gctcgtggcc cggcaggagg
     1141 actccggact ccgggatcac agtgtcaggg tcctcatttc caaccatgtg acacctttcg
     1201 accacaacat agtcaatttg cttaccacct gtagcacccc tctactcaat agtcccccca
     1261 gctttgtgtg ctggtctcgg ggcttcatgg agatgaatgg gcggggggag ttggtggagt
     1321 cactcaagag attctgtgct tccacgaggc ttccccccac tcctctgctg ctattccctg
     1381 aggaagaggc caccaatggc cgggaggggc tcctgcgctt cagttcctgg ccattttcta
     1441 tccaagatgt ggtacaacct cttaccctgc aagttcagag acccctggtc tctgtgacgg
     1501 tgtcagatgc ctcctgggtc tcagaactgc tgtggtcact tttcgtccct ttcacggtgt
     1561 atcaagtaag gtggcttcgt cctgttcatc gccaactagg ggaagcgaat gaggagtttg
     1621 cactccgtgt acaacagctg gtggccaagg aattgggcca gacagggaca cggctcactc
     1681 cagctgacaa agcagagcac atgaagcgac aaagacaccc cagattgcgc ccccagtcag
     1741 cccagtcttc tttccctccc tcccctggtc cttctcctga tgtgcaactg gcaactctgg
     1801 ctcagagagt caaggaagtt ttgccccatg tgccattggg tgtcatccag agagacctgg
     1861 ccaagactgg ctgtgtagac ttgactatca ctaatctgct tgagggggcc gtagctttca
     1921 tgcctgaaga catcaccaag ggaactcagt ccctacccac agcctctgcc tccaagtttc
     1981 ccagctctgg cccggtgacc cctcagccaa cagccctaac atttgccaag tcttcctggg
     2041 cccggcagga gagcctgcag gagcgcaagc aagcactata tgaatacgca agaaggagat
     2101 tcacagagag acgagcccag gaggctgacc tcgaggtcga ggactacaag gacgacgatg
     2161 acaagatctt cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     2221 aggaagctga gttggctgct ctagagggcc ctattctata gtgtcaccta aatgctagag
     2281 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2341 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2401 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2461 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2521 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2581 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2641 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2701 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2761 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2821 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2881 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2941 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3001 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3061 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3121 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3181 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3241 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3301 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3361 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3421 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3481 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3541 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3601 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3661 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3721 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3781 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3841 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3901 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     3961 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4021 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4081 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4141 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4201 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4261 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4321 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4381 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4441 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4501 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4561 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4621 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4681 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4741 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4801 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4861 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4921 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4981 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5041 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5101 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5161 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5221 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5281 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5341 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5401 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5461 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5521 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5581 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5641 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5701 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5761 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5821 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5881 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5941 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6001 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6061 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6121 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6181 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6241 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6301 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6361 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6421 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6481 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6541 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6601 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6661 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     6721 ccgatcccct atggtgcact ctcagt
//
