LOCUS       sequence-383994-        6746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Assessing G2BR function in 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 A397D
FEATURES             Location/Qualifiers
     source          1..6746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        185..564
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        565..768
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     719..739
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     813..832
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        813..831
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2136..2159
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2169..2186
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2249..2267)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2250..2267)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2287..2304)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2293..2517
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2563..2991
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2650..2669)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2860..2881
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3000..3020)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3005..3334
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3185..3320
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3247..3266
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3401..4195
                     /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(3455..3474)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4065..4084
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4369..4490
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4406..4425)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4460..4479
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4539..4555)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4539..4555)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4552..4574)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4563..4579
                     /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(4587..4593,4594..4611,4612..4617))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4632..4653
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4770..4787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4941..5529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5021..5040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5700..6491,6492..6560))
                     /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     6323..6342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6561..6665)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(6740..6746,1..13))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
       61 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      121 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      181 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      241 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      301 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      361 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      421 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      481 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      541 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      601 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      661 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      721 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      781 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      841 gcttggtacc gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcatggagc
      901 ttccctcagg gccggggccg gagcggctct ttgactcgca ccggcttccg ggtgactgct
      961 tcctactgct cgtgctgctg ctctacgcgc cagtcgggtt ctgcctcctc gtcctgcgcc
     1021 tctttctcgg gatccacgtc ttcctggtca gctgcgcgct gccagacagc gtccttcgca
     1081 gattcgtagt gcggaccatg tgtgcggtgc tagggctcgt ggcccggcag gaggactccg
     1141 gactccggga tcacagtgtc agggtcctca tttccaacca tgtgacacct ttcgaccaca
     1201 acatagtcaa tttgcttacc acctgtagca cccctctact caatagtccc cccagctttg
     1261 tgtgctggtc tcggggcttc atggagatga atgggcgggg ggagttggtg gagtcactca
     1321 agagattctg tgcttccacg aggcttcccc ccactcctct gctgctattc cctgaggaag
     1381 aggccaccaa tggccgggag gggctcctgc gcttcagttc ctggccattt tctatccaag
     1441 atgtggtaca acctcttacc ctgcaagttc agagacccct ggtctctgtg acggtgtcag
     1501 atgcctcctg ggtctcagaa ctgctgtggt cacttttcgt ccctttcacg gtgtatcaag
     1561 taaggtggct tcgtcctgtt catcgccaac taggggaagc gaatgaggag tttgcactcc
     1621 gtgtacaaca gctggtggcc aaggaattgg gccagacagg gacacggctc actccagctg
     1681 acaaagcaga gcacatgaag cgacaaagac accccagatt gcgcccccag tcagcccagt
     1741 cttctttccc tccctcccct ggtccttctc ctgatgtgca actggcaact ctggctcaga
     1801 gagtcaagga agttttgccc catgtgccat tgggtgtcat ccagagagac ctggccaaga
     1861 ctggctgtgt agacttgact atcactaatc tgcttgaggg ggccgtagct ttcatgcctg
     1921 aagacatcac caagggaact cagtccctac ccacagcctc tgcctccaag tttcccagct
     1981 ctggcccggt gacccctcag ccaacagccc taacatttgc caagtcttcc tgggcccggc
     2041 aggagagcct gcaggagcgc aagcaagcac tatatgaata cgacagaagg agattcacag
     2101 agagacgagc ccaggaggct gacctcgagg tcgaggacta caaggacgac gatgacaaga
     2161 tcttcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     2221 ctgagttggc tgctctagag ggccctattc tatagtgtca cctaaatgct agagctcgct
     2281 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2341 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2401 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2461 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     2521 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     2581 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2641 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2701 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2761 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2821 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2881 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2941 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     3001 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3061 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3121 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3181 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3241 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     3301 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     3361 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3421 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3481 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3541 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3601 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3661 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3721 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3781 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3841 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3901 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     3961 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     4021 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4081 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4141 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     4201 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     4261 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4321 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4381 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4441 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4501 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4561 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4621 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4681 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4741 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4801 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4861 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4921 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4981 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5041 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5101 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5161 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5221 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5281 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5341 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5401 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5461 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5521 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5581 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5641 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5701 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5761 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5821 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5881 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5941 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6001 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6061 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6121 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6181 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6241 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6301 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6361 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6421 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6481 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6541 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6601 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6661 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     6721 ccctatggtg cactctcagt acaatc
//
