LOCUS       sequence-7149-ex        6828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6828)
  AUTHORS   Heessen S, Dantuma NP, Tessarz P, Jellne M, Masucci MG
  TITLE     Inhibition of ubiquitin/proteasome-dependent proteolysis in 
            Saccharomyces cerevisiae by a Gly-Ala repeat.
  JOURNAL   FEBS Lett. 2003 Dec 4. 555(2):397-404.
  PUBMED    14644450
REFERENCE   2  (bases 1 to 6828)
  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: "FEBS Lett. 
            2003 Dec 4. 555(2):397-404."
COMMENT     Sequence Label: pYES2-Ub-M-GFP
FEATURES             Location/Qualifiers
     source          1..6828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..444
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2..119
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     415..438
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     476..495
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        476..494
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             560..787
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     regulatory      821..830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(827..829,830..832,833..1546)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(827..829,830..832,833..1543)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(872..893)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1133..1152)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1480..1501
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      1581..1828
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1597..1615)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1898..1915)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2069..2657)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2149..2168)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2828..3619,3620..3688))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLID*MEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     CDS             complement(3784..4587)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4588..4808)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     rep_origin      5407..6287
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     rep_origin      complement(6356..6811)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     6705..6724
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt
       61 cctcgtcctc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga
      121 acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac
      181 ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga
      241 ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat
      301 taacagatat ataaatgcaa aaactgcatt aaccacttta actaatactt tcaacatttt
      361 cggtttgtat tacttcttat tcaaatgtaa taaaagtatc aacaaaaaat tgttaatata
      421 cctctatact ttaacgtcaa ggagaaaaaa ccccggatcg gactactagc agctgtaata
      481 cgactcacta tagggaatat taagcttggt accgagctcg gatccactag taacggccgc
      541 cagtgtgctg gaattcacca tgcagatctt cgtgaagact ctgactggta agaccatcac
      601 cctcgaggtt gagcccagtg acaccattga gaatgtcaag gcaaagatcc aagataagga
      661 aggcatccct cctgaccagc agaggctgat ctttgctgga aaacagctgg aagatgggcg
      721 caccctgtct gactacaaca tccagaaaga gtccaccctg cacctggtac tccgtctcag
      781 aggtgggatg gggaagcttg gtcgacagga tccaccggtc gccaccatgg tgagcaaggg
      841 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      901 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      961 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1021 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1081 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1141 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1201 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1261 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1321 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1381 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1441 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1501 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg gccgctcgag
     1561 catgcatcta gagggccgca tcatgtaatt agttatgtca cgcttacatt cacgccctcc
     1621 ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat
     1681 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt
     1741 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt
     1801 tttgggacgc tcgaaggctt taatttgcgg ccctgcatta atgaatcggc caacgcgcgg
     1861 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1921 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1981 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aagcccagga
     2041 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2101 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2161 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2221 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2281 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2341 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2401 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2461 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     2521 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2581 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2641 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2701 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2761 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2821 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2881 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag cgcttaccat
     2941 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3001 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3061 ccattcagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3121 tgcgcaacgt tgttggcatt gctacaggca tcgtggtgtc actctcgtcg tttggtatgg
     3181 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3241 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3301 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3361 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3421 cgagttgctc ttgcccggcg tcaatacggg ataatagtgt atcacatagc agaactttaa
     3481 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3541 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3601 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3661 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatgggta ataactgata
     3721 taattaaatt gaagctctaa tttgtgagtt tagtatacat gcatttactt ataatacagt
     3781 tttttagttt tgctggccgc atcttctcaa atatgcttcc cagcctgctt ttctgtaacg
     3841 ttcaccctct accttagcat cccttccctt tgcaaatagt cctcttccaa caataataat
     3901 gtcagatcct gtagagacca catcatccac ggttctatac tgttgaccca atgcgtctcc
     3961 cttgtcatct aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc
     4021 acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc
     4081 aacagtaccc ttagtatatt ctccagtaga tagggagccc ttgcatgaca attctgctaa
     4141 catcaaaagg cctctaggtt cctttgttac ttcttctgcc gcctgcttca aaccgctaac
     4201 aatacctggg cccaccacac cgtgtgcatt cgtaatgtct gcccattctg ctattctgta
     4261 tacacccgca gagtactgca atttgactgt attaccaatg tcagcaaatt ttctgtcttc
     4321 gaagagtaaa aaattgtact tggcggataa tgcctttagc ggcttaactg tgccctccat
     4381 ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa caaattttgg gacctaatgc
     4441 ttcaactaac tccagtaatt ccttggtggt acgaacatcc aatgaagcac acaagtttgt
     4501 ttgcttttcg tgcatgatat taaatagctt ggcagcaaca ggactaggat gagtagcagc
     4561 acgttcctta tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg tttttgttct
     4621 gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacactac atatgcgtat
     4681 atataccaat ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg agattaccga
     4741 atcaaaaaaa tttcaaagaa accgaaatca aaaaaaagaa taaaaaaaaa atgatgaatt
     4801 gaattgaaaa gctagcttat cgatgataag ctgtcaaaga tgagaattaa ttccacggac
     4861 tatagactat actagatact ccgtctactg tacgatacac ttccgctcag gtccttgtcc
     4921 tttaacgagg ccttaccact cttttgttac tctattgatc cagctcagca aaggcagtgt
     4981 gatctaagat tctatcttcg cgatgtagta aaactagcta gaccgagaaa gagactagaa
     5041 atgcaaaagg cacttctaca atggctgcca tcattattat ccgatgtgac gctgcagctt
     5101 ctcaatgata ttcgaatacg ctttgaggag atacagccta atatccgaca aactgtttta
     5161 cagatttacg atcgtacttg ttacccatca ttgaattttg aacatccgaa cctgggagtt
     5221 ttccctgaaa cagatagtat atttgaacct gtataataat atatagtcta gcgctttacg
     5281 gaagacaatg tatgtatttc ggttcctgga gaaactattg catctattgc ataggtaatc
     5341 ttgcacgtcg catccccggt tcattttctg cgtttccatc ttgcacttca atagcatatc
     5401 tttgttaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta
     5461 atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg
     5521 ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc aacgcgacga
     5581 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg
     5641 cgagagcgct attttaccaa caaagaatct atacttcttt tttgttctac aaaaatgcat
     5701 cccgagagcg ctatttttct aacaaagcat cttagattac tttttttctc ctttgtgcgc
     5761 tctataatgc agtctcttga taactttttg cactgtaggt ccgttaaggt tagaagaagg
     5821 ctactttggt gtctattttc tcttccataa aaaaagcctg actccacttc ccgcgtttac
     5881 tgattactag cgaagctgcg ggtgcatttt ttcaagataa aggcatcccc gattatattc
     5941 tataccgatg tggattgcgc atactttgtg aacagaaagt gatagcgttg atgattcttc
     6001 attggtcaga aaattatgaa cggtttcttc tattttgtct ctatatacta cgtataggaa
     6061 atgtttacat tttcgtattg ttttcgattc actctatgaa tagttcttac tacaattttt
     6121 ttgtctaaag agtaatacta gagataaaca taaaaaatgt agaggtcgag tttagatgca
     6181 agttcaagga gcgaaaggtg gatgggtagg ttatataggg atatagcaca gagatatata
     6241 gcaaagagat acttttgagc aatgtttgtg gaagcggtat tcgcaatggg aagctccacc
     6301 ccggttgata atcagaaaag ccccaaaaac aggaagattg tataagcaaa tatttaaatt
     6361 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     6421 aacgaatagc ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     6481 ttgagtgttg ttccagtttc caacaagagt ccactattaa agaacgtgga ctccaacgtc
     6541 aaagggcgaa aaagggtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     6601 agttttttgg ggtcgaggtg ccgtaaagca gtaaatcgga agggtaaacg gatgccccca
     6661 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     6721 ggagcggggg ctagggcggt gggaagtgta ggggtcacgc tgggcgtaac caccacaccc
     6781 gccgcgctta atggggcgct acagggcgcg tggggatgat ccactagt
//
