LOCUS       sequence-191759-        6691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast episomal vector for expression of His-tagged Smt3 (SUMO); LEU2
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6691)
  AUTHORS   Ulrich HD, Davies AA
  TITLE     In vivo detection and characterization of sumoylation targets in 
            Saccharomyces cerevisiae.
  JOURNAL   Methods Mol Biol. 2009;497:81-103. doi: 10.1007/978-1-59745-566-4_6.
  PUBMED    19107412
REFERENCE   2  (bases 1 to 6691)
  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: "Methods Mol
            Biol."; date: "2009"; volume: "497:81-103. doi"; pages: " 
            10.1007/978-1-59745-566-4_6"
COMMENT     Sequence Label: YEp181-CUP1-His-Smt3
FEATURES             Location/Qualifiers
     source          1..6691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..235
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             282..302
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             309..602
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     terminator      636..823
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(691..715)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(847..863)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(847..863)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(860..882)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    871..887
                     /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(895..901,902..919,920..925))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    940..961
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1078..1095)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1249..1837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1329..1348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2008..2799,2800..2868))
                     /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     2631..2650
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2869..2973)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3041..3059
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3079..3484
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3485..4579
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     rep_origin      4717..6063
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     6485..6507
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6499..6516
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6500..6516
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ctagttagaa aaagacattt ttgctgtcag tcactgtcaa gagattcttt tgctggcatt
       61 tcttctagaa gcaaaaagag cgatgcgtct tttccgctga accgttccag caaaaaagac
      121 taccaacgca atatggattg tcagaatcat ataaaagaga agcaaataac tccttgtctt
      181 gtatcaattg cattataata tcttcttgtt agtgcaatat catatagaag tcatcgaaat
      241 agatattaag aaaaacaaac tgtaacggat ccattatggg ccatcatcac catcatcacc
      301 atagcggcat gtcggactca gaagtcaatc aagaagctaa gccagaggtc aagccagaag
      361 tcaagcctga gactcacatc aatttaaagg tgtccgatgg atcttcagag atcttcttca
      421 agatcaaaaa gaccactcct ttaagaaggc tgatggaagc gttcgctaaa agacagggta
      481 aggaaatgga ctccttaaga ttcttgtacg acggtattag aattcaagct gatcagaccc
      541 ctgaagattt ggacatggag gataacgata ttattgaggc tcacagagaa cagattggtg
      601 gtgctacgta ttagctgcag ccaagctaat tccgggcgaa tttcttatga tttatgattt
      661 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg
      721 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc
      781 aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgcaa gcttggcgta
      841 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
      901 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
      961 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     1021 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     1081 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1141 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1201 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1261 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1321 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1381 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1441 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1501 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1561 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1621 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1681 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1741 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     1801 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     1861 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     1921 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     1981 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2041 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2101 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2161 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2221 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2281 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     2341 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2401 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2461 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2521 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2581 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     2641 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     2701 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     2761 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     2821 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     2881 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2941 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     3001 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     3061 ctttcgtctt caagaattaa ctgtgggaat actcaggtat cgtaagatgc aagagttcga
     3121 atctcttagc aaccattatt tttttcctca acataacgag aacacacagg ggcgctatcg
     3181 cacagaatca aattcgatga ctggaaattt tttgttaatt tcagaggtcg cctgacgcat
     3241 ataccttttt caactgaaaa attgggagaa aaaggaaagg tgagagcgcc ggaaccggct
     3301 tttcatatag aatagagaag cgttcatgac taaatgcttg catcacaata cttgaagttg
     3361 acaatattat ttaaggacct attgtttttt ccaataggtg gttagcaatc gtcttacttt
     3421 ctaacttttc ttacctttta catttcagca atatatatat atatttcaag gatataccat
     3481 tctaatgtct gcccctaaga agatcgtcgt tttgccaggt gaccacgttg gtcaagaaat
     3541 cacagccgaa gccattaagg ttcttaaagc tatttctgat gttcgttcca atgtcaagtt
     3601 cgatttcgaa aatcatttaa ttggtggtgc tgctatcgat gctacaggtg ttccacttcc
     3661 agatgaggcg ctggaagcct ccaagaaggc tgatgccgtt ttgttaggtg ctgtgggtgg
     3721 tcctaaatgg ggtacaggta gtgttagacc tgaacaaggt ttactaaaaa tccgtaaaga
     3781 acttcaattg tacgccaact taagaccatg taactttgca tccgactctc ttttagactt
     3841 atctccaatc aagccacaat ttgctaaagg tactgacttc gttgttgtca gagaattagt
     3901 gggaggtatt tactttggta agagaaagga agacgatggt gatggtgtcg cttgggatag
     3961 tgaacaatac accgttccag aagtgcaaag aatcacaaga atggccgctt tcatggccct
     4021 acaacatgag ccaccattgc ctatttggtc cttggataaa gctaatgttt tggcctcttc
     4081 aagattatgg agaaaaactg tggaggaaac catcaagaac gaatttccta cattgaaggt
     4141 tcaacatcaa ttgattgatt ctgccgccat gatcctagtt aagaacccaa cccacctaaa
     4201 tggtattata atcaccagca acatgtttgg tgatatcatc tccgatgaag cctccgttat
     4261 cccaggttcc ttgggtttgt tgccatctgc gtccttggcc tctttgccag acaagaacac
     4321 cgcatttggt ttgtacgaac catgccacgg ttctgctcca gatttgccaa agaataaggt
     4381 caaccctatc gccactatct tgtctgctgc aatgatgttg aaattgtcat tgaacttgcc
     4441 tgaagaaggt aaggccattg aagatgcagt taaaaaggtt ttggatgcag gtatcagaac
     4501 tggtgattta ggtggttcca acagtaccac cgaagtcggt gatgctgtcg ccgaagaagt
     4561 taagaaaatc cttgcttaaa aagattctct ttttttatga tatttgtaca taaactttat
     4621 aaatgaaatt cataatagaa acgacacgaa attacaaaat ggaatatgtt catagggtag
     4681 aattaattct catgtttgac agcttatcat cggatcgatc caatatcaaa ggaaatgata
     4741 gcattgaagg atgagactaa tccaattgag gagtggcagc atatagaaca gctaaagggt
     4801 agtgctgaag gaagcatacg ataccccgca tggaatggga taatatcaca ggaggtacta
     4861 gactaccttt catcctacat aaatagacgc atataagtac gcatttaagc ataaacacgc
     4921 actatgccgt tcttctcatg tatatatata tacaggcaac acgcagatat aggtgcgacg
     4981 tgaacagtga gctgtatgtg cgcagctcgc gttgcatttt cggaagcgct cgttttcgga
     5041 aacgctttga agttcctatt ccgaagttcc tattctctag ctagaaagta taggaacttc
     5101 agagcgcttt tgaaaaccaa aagcgctctg aagacgcact ttcaaaaaac caaaaacgca
     5161 ccggactgta acgagctact aaaatattgc gaataccgct tccacaaaca ttgctcaaaa
     5221 gtatctcttt gctatatatc tctgtgctat atccctatat aacctaccca tccacctttc
     5281 gctccttgaa cttgcatcta aactcgacct ctacattttt tatgtttatc tctagtatta
     5341 ctctttagac aaaaaaattg tagtaagaac tattcataga gtgaatcgaa aacaatacga
     5401 aaatgtaaac atttcctata cgtagtatat agagacaaaa tagaagaaac cgttcataat
     5461 tttctgacca atgaagaatc atcaacgcta tcactttctg ttcacaaagt atgcgcaatc
     5521 cacatcggta tagaatataa tcggggatgc ctttatcttg aaaaaatgca cccgcagctt
     5581 cgctagtaat cagtaaacgc gggaagtgga gtcaggcttt ttttatggaa gagaaaatag
     5641 acaccaaagt agccttcttc taaccttaac ggacctacag tgcaaaaagt tatcaagaga
     5701 ctgcattata gagcgcacaa aggagaaaaa aagtaatcta agatgctttg ttagaaaaat
     5761 agcgctctcg ggatgcattt ttgtagaaca aaaaagaagt atagattctt tgttggtaaa
     5821 atagcgctct cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa
     5881 aaattagcgc tctcgcgttg catttttgtt ttacaaaaat gaagcacaga ttcttcgttg
     5941 gtaaaatagc gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt
     6001 ttgaaaaatt agcgctctcg cgttgcattt ttgttctaca aaatgaagca cagatgcttc
     6061 gttaacaaag atatgctatt gaagtgcaag atggaaacgc agaaaatgaa ccggggatgc
     6121 gacgtgcaag attacctatg caatagatgc aatagtttct ccaggaaccg aaatacatac
     6181 attgtcttcc gtaaagcgct agactatata ttattataca ggttcaaata tactatctgt
     6241 ttcagggaaa actcccaggt tcggatgttc aaaattcaat gatgggtaac aagtacgatc
     6301 cgataaatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     6361 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     6421 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     6481 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tcgagctccc attaccgaca
     6541 tttgggcgct atacgtgcat atgttcatgt atgtatctgt atttaaaaca cttttgtatt
     6601 atttttcctc atatatgtgt ataggtttat acggatgatt taattattac ttcaccaccc
     6661 tttatttcag gctgatatct tagccttgtt a
//
