LOCUS       sequence-12742-e        6476 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 6476)
  AUTHORS   Tagwerker C, Flick K, Cui M, Guerrero C, Dou Y, Auer B, Baldi P, 
            Huang L, Kaiser P
  TITLE     A tandem affinity tag for two-step purification under fully 
            denaturing conditions: application in ubiquitin profiling and 
            protein complex identification combined with in vivocross-linking.
  JOURNAL   Mol Cell Proteomics. 2006 Apr . 5(4):737-48.
  PUBMED    16432255
REFERENCE   2  (bases 1 to 6476)
  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: "Mol Cell 
            Proteomics. 2006 Apr . 5(4):737-48."
COMMENT     Sequence Label: YEpURA-CUP1-HBTubiquitin
FEATURES             Location/Qualifiers
     source          1..6476
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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)."
     primer_bind     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        412..646
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             724..741
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             994..1014
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1060..1284
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="QIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFA
                     GKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(1338..1356)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1525..1542)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1525..1541)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1534..1556)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1978..3324
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(3435..4238)
                     /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(4239..4459)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4484..4502)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4570..4674
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4675..4743,4744..5535)
                     /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     complement(4893..4912)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5706..6294
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6195..6214
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6448..6465
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttta
      241 agccgatccc attaccgaca tttgggcgct atacgtgcat atgttcatgt atgtatctgt
      301 atttaaaaca cttttgtatt atttttcctc atatatgtgt ataggtttat acggatgatt
      361 taattattac ttcaccaccc tttatttcag gctgatatct tagccttgtt actagttaga
      421 aaaagacatt tttgctgtca gtcactgtca agagattctt ttgctggcat ttcttctaga
      481 agcaaaaaga gcgatgcgtc ttttccgctg aaccgttcca gcaaaaaaga ctaccaacgc
      541 aatatggatt gtcagaatca tataaaagag aagcaaataa ctccttgtct tgtatcaatt
      601 gcattataat atcttcttgt tagtgcaata tcatatagaa gtcatcgaaa tagatattaa
      661 gaaaaacaaa ctgtacaatc aatcaatcaa tcatcacata aaggatccat tatgaggggt
      721 tcacatcatc accaccatca tgctggaaag gccggtgaag gtgaaatccc tgcccctctt
      781 gctggtaccg tttctaagat actggtaaaa gaaggtgaca ctgttaaagc tggtcaaaca
      841 gttctggtgc tggaggctat gaaaatggag acagaaatta acgctcctac tgacggaaaa
      901 gttgaaaagg tgttagttaa ggaaagagat gctgttcaag gtggtcaagg tctaatcaag
      961 atcggcgact acgatatacc cacaaccgcc agcgagaatc tgtactttca atcagagtta
     1021 aaaaccgccg cactagctca acatgacgag gcttctagac agattttcgt caagactttg
     1081 accggtaaaa ccataacatt ggaagttgaa tcttccgata ccatcgacaa cgttaagtcg
     1141 aaaattcaag acaaggaagg tatccctcca gatcaacaaa gattgatctt tgccggtaag
     1201 cagctagaag acggtagaac gctgtctgat tacaacattc agaaggagtc caccttacat
     1261 cttgtcttaa gactaagagg tggttgagga tcctaaacag gccccttttc ctttgtcgat
     1321 atcatgtaat tagttatgtc acgcttacat tcacgccctc ctcccacatc cgctctaacc
     1381 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt ttaatagtta
     1441 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacaaacgcg
     1501 tgtacgcatg taacattaga attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     1561 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     1621 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     1681 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatcg
     1741 gatcgtactt gttacccatc attgaatttt gaacatccga acctgggagt tttccctgaa
     1801 acagatagta tatttgaacc tgtataataa tatatagtct agcgctttac ggaagacaat
     1861 gtatgtattt cggttcctgg agaaactatt gcatctattg cataggtaat cttgcacgtc
     1921 gcatccccgg ttcattttct gcgtttccat cttgcacttc aatagcatat ctttgttaac
     1981 gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa
     2041 acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac
     2101 caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt
     2161 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat
     2221 tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct
     2281 atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag
     2341 tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt
     2401 ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg
     2461 aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg
     2521 gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa
     2581 attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt
     2641 tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag
     2701 taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc
     2761 gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac
     2821 ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg
     2881 tgcgtttttg gttttttgaa agtgcgtctt cagagcgctt ttggttttca aaagcgctct
     2941 gaagttccta tactttctag ctagagaata ggaacttcgg aataggaact tcaaagcgtt
     3001 tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg cacatacagc tcactgttca
     3061 cgtcgcacct atatctgcgt gttgcctgta tatatatata catgagaaga acggcatagt
     3121 gcgtgtttat gcttaaatgc gtacttatat gcgtctattt atgtaggatg aaaggtagtc
     3181 tagtacctcc tgtgatatta tcccattcca tgcggggtat cgtatgcttc cttcagcact
     3241 accctttagc tgttctatat gctgccactc ctcaattgga ttagtctcat ccttcaatgc
     3301 tatcatttcc tttgatattg gatcgatccg atgataagct gtcaaacatg agaattgggt
     3361 aataactgat ataattaaat tgaagctcta atttgtgagt ttagtataca tgcatttact
     3421 tataatacag ttttttagtt ttgctggccg catcttctca aatatgcttc ccagcctgct
     3481 tttctgtaac gttcaccctc taccttagca tcccttccct ttgcaaatag tcctcttcca
     3541 acaataataa tgtcagatcc tgtagagacc acatcatcca cggttctata ctgttgaccc
     3601 aatgcgtctc ccttgtcatc taaacccaca ccgggtgtca taatcaacca atcgtaacct
     3661 tcatctcttc cacccatgtc tctttgagca ataaagccga taacaaaatc tttgtcgctc
     3721 ttcgcaatgt caacagtacc cttagtatat tctccagtag atagggagcc cttgcatgac
     3781 aattctgcta acatcaaaag gcctctaggt tcctttgtta cttcttctgc cgcctgcttc
     3841 aaaccgctaa caatacctgg gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct
     3901 gctattctgt atacacccgc agagtactgc aatttgactg tattaccaat gtcagcaaat
     3961 tttctgtctt cgaagagtaa aaaattgtac ttggcggata atgcctttag cggcttaact
     4021 gtgccctcca tggaaaaatc agtcaagata tccacatgtg tttttagtaa acaaattttg
     4081 ggacctaatg cttcaactaa ctccagtaat tccttggtgg tacgaacatc caatgaagca
     4141 cacaagtttg tttgcttttc gtgcatgata ttaaatagct tggcagcaac aggactagga
     4201 tgagtagcag cacgttcctt atatgtagct ttcgacatga tttatcttcg tttcctgcat
     4261 gtttttgttc tgtgcagttg ggttaagaat actgggcaat ttcatgtttc ttcaacacta
     4321 catatgcgta tatataccaa tctaagtctg tgctccttcc ttcgttcttc cttctgttcg
     4381 gagattaccg aatcaaaaaa atttcaaaga aaccgaaatc aaaaaaaaga ataaaaaaaa
     4441 aatgatgaat tgaattgaaa agctaattct tgaagacgaa agggcctcgt gatacgccta
     4501 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4561 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4621 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4681 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4741 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4801 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4861 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4921 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4981 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5041 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5101 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5161 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5221 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5281 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5341 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5401 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5461 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5521 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5581 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5641 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5701 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5761 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5821 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     5881 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5941 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6001 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6061 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6121 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6181 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6241 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6301 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6361 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     6421 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaga
//
