LOCUS       Exported                6187 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Yeast episomal vector for expression of His-tagged Smt3 (SUMO); URA3
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    YEp195-CUP1-His-Smt3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6187)
  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 6187)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6187
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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(895..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(2008..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        3084..3299
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3300..4103
                     /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"
     rep_origin      4214..5560
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     5981..6003
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5995..6012
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5996..6012
                     /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 caagaattag cttttcaatt catcattttt tttttattct tttttttgat
     3121 ttcggtttcc ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga
     3181 aggagcacag acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt
     3241 gcccagtatt cttaacccaa ctgcacagaa caaaaacatg caggaaacga agataaatca
     3301 tgtcgaaagc tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc
     3361 tatttaatat catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca
     3421 ccaaggaatt actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac
     3481 atgtggatat cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat
     3541 ccgccaagta caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag
     3601 tcaaattgca gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg
     3661 cacacggtgt ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa
     3721 caaaggaacc tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta
     3781 ctggagaata tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg
     3841 gctttattgc tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga
     3901 cacccggtgt gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg
     3961 atgatgtggt ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg
     4021 gaagggatgc taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga
     4081 gaagatgcgg ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact
     4141 cacaaattag agcttcaatt taattatatc agttattacc caattctcat gtttgacagc
     4201 ttatcatcgg atcgatccaa tatcaaagga aatgatagca ttgaaggatg agactaatcc
     4261 aattgaggag tggcagcata tagaacagct aaagggtagt gctgaaggaa gcatacgata
     4321 ccccgcatgg aatgggataa tatcacagga ggtactagac tacctttcat cctacataaa
     4381 tagacgcata taagtacgca tttaagcata aacacgcact atgccgttct tctcatgtat
     4441 atatatatac aggcaacacg cagatatagg tgcgacgtga acagtgagct gtatgtgcgc
     4501 agctcgcgtt gcattttcgg aagcgctcgt tttcggaaac gctttgaagt tcctattccg
     4561 aagttcctat tctctagcta gaaagtatag gaacttcaga gcgcttttga aaaccaaaag
     4621 cgctctgaag acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa
     4681 atattgcgaa taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct
     4741 gtgctatatc cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac
     4801 tcgacctcta cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag
     4861 taagaactat tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt
     4921 agtatataga gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc
     4981 aacgctatca ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg
     5041 gggatgcctt tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg
     5101 aagtggagtc aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa
     5161 ccttaacgga cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg
     5221 agaaaaaaag taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg
     5281 tagaacaaaa aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc
     5341 tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat
     5401 ttttgtttta caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc
     5461 atttctgttc tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt
     5521 tgcatttttg ttctacaaaa tgaagcacag atgcttcgtt aacaaagata tgctattgaa
     5581 gtgcaagatg gaaacgcaga aaatgaaccg gggatgcgac gtgcaagatt acctatgcaa
     5641 tagatgcaat agtttctcca ggaaccgaaa tacatacatt gtcttccgta aagcgctaga
     5701 ctatatatta ttatacaggt tcaaatatac tatctgtttc agggaaaact cccaggttcg
     5761 gatgttcaaa attcaatgat gggtaacaag tacgatccga ttatgcggtg tgaaataccg
     5821 cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac
     5881 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     5941 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     6001 acgacggcca gtgaattcga gctcccatta ccgacatttg ggcgctatac gtgcatatgt
     6061 tcatgtatgt atctgtattt aaaacacttt tgtattattt ttcctcatat atgtgtatag
     6121 gtttatacgg atgatttaat tattacttca ccacccttta tttcaggctg atatcttagc
     6181 cttgtta
//
