LOCUS       sequence-384515-        6547 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Assessing domain requirements of AUP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6547)
  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 6547)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 DTM
FEATURES             Location/Qualifiers
     source          1..6547
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        183..562
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        563..766
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     717..737
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     811..830
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        811..829
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      861..870
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1935..1958
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1968..1985
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2048..2066)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2049..2066)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2086..2103)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2092..2316
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2362..2790
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2449..2468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2659..2680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2799..2819)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2804..3133
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2984..3119
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3046..3065
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3200..3994
                     /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(3254..3273)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3864..3883
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4168..4289
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4205..4224)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4259..4278
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4338..4354)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4338..4354)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4351..4373)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4362..4378
                     /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(4386..4392,4393..4410,4411..4416))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4431..4452
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4569..4586)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4740..5328)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4820..4839)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5499..6290,6291..6359))
                     /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     6122..6141
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6360..6464)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(6539..6547,1..11))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
       61 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
      121 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      181 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      241 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      301 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      361 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      421 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      481 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      541 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      601 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      661 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      721 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
      781 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
      841 ttggtaccga gctcggatcc gccaccatgg tccttcgcag attcgtagtg cggaccatgt
      901 gtgcggtgct agggctcgtg gcccggcagg aggactccgg actccgggat cacagtgtca
      961 gggtcctcat ttccaaccat gtgacacctt tcgaccacaa catagtcaat ttgcttacca
     1021 cctgtagcac ccctctactc aatagtcccc ccagctttgt gtgctggtct cggggcttca
     1081 tggagatgaa tgggcggggg gagttggtgg agtcactcaa gagattctgt gcttccacga
     1141 ggcttccccc cactcctctg ctgctattcc ctgaggaaga ggccaccaat ggccgggagg
     1201 ggctcctgcg cttcagttcc tggccatttt ctatccaaga tgtggtacaa cctcttaccc
     1261 tgcaagttca gagacccctg gtctctgtga cggtgtcaga tgcctcctgg gtctcagaac
     1321 tgctgtggtc acttttcgtc cctttcacgg tgtatcaagt aaggtggctt cgtcctgttc
     1381 atcgccaact aggggaagcg aatgaggagt ttgcactccg tgtacaacag ctggtggcca
     1441 aggaattggg ccagacaggg acacggctca ctccagctga caaagcagag cacatgaagc
     1501 gacaaagaca ccccagattg cgcccccagt cagcccagtc ttctttccct ccctcccctg
     1561 gtccttctcc tgatgtgcaa ctggcaactc tggctcagag agtcaaggaa gttttgcccc
     1621 atgtgccatt gggtgtcatc cagagagacc tggccaagac tggctgtgta gacttgacta
     1681 tcactaatct gcttgagggg gccgtagctt tcatgcctga agacatcacc aagggaactc
     1741 agtccctacc cacagcctct gcctccaagt ttcccagctc tggcccggtg acccctcagc
     1801 caacagccct aacatttgcc aagtcttcct gggcccggca ggagagcctg caggagcgca
     1861 agcaagcact atatgaatac gcaagaagga gattcacaga gagacgagcc caggaggctg
     1921 acctcgaggt cgaggactac aaggacgacg atgacaagat cttcgagcac caccaccacc
     1981 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctctagagg
     2041 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     2101 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2161 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2221 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2281 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     2341 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2401 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2461 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2521 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2581 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2641 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2701 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2761 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     2821 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2881 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2941 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3001 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3061 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3121 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     3181 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3241 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3301 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     3361 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     3421 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     3481 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     3541 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     3601 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     3661 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     3721 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     3781 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     3841 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     3901 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     3961 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4021 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4081 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4141 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4201 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4261 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4321 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4381 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4441 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4501 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4561 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4621 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4681 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4741 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4801 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4861 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4921 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4981 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5041 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5101 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5161 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5221 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5281 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5341 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5401 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5461 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5521 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5581 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5641 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5701 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5761 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5821 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5881 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5941 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6001 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6061 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6121 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6181 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6241 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6301 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6361 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6421 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6481 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     6541 caatctg
//
