LOCUS       sequence-420583-        4927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal PCR-based tagging with yeGFP-His6 in budding yeast with 
            HygR selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4927)
  AUTHORS   Renz C, Asimaki E, Meister C, Albanese V, Petriukov K, Krapoth NC, 
            Wegmann S, Wollscheid HP, Wong RP, Fulzele A, Chen JX, Leon S, 
            Ulrich HD
  TITLE     Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.
  JOURNAL   Mol Cell. 2024 Jan 18;84(2):386-400.e11. doi: 
            10.1016/j.molcel.2023.11.016. Epub 2023 Dec 15.
  PUBMED    38103558
REFERENCE   2  (bases 1 to 4927)
  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"; doi: 
            "10.1016/j.molcel.2023.11.016"; journalName: "Mol Cell"; date: 
            "2024-01-18- 18"; volume: "84"; issue: "2"; pages: "386-400.e11"
COMMENT     Sequence Label: pHyg-yeGFP-His6
FEATURES             Location/Qualifiers
     source          1..4927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             85..795
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             802..819
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        875..1218
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1219..2247
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1219..2247
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2255..2502
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2271..2289)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2564..2583)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2565..2583)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2670..2687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2841..3429)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2921..2940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3600..4391,4392..4460))
                     /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     4223..4242
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4461..4565)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4633..4651
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4689..4711)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4811..4830
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4911..4927,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4911..4927,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatccgg agcaggtgct
       61 ggtgctggtg ctggagcaat tctgtctaaa ggtgaagaat tattcactgg tgttgtccca
      121 attttggttg aattagatgg tgatgttaat ggtcacaaat tttctgtctc cggtgaaggt
      181 gaaggtgatg ctacttacgg taaattgacc ttaaaattta tttgtactac tggtaaattg
      241 ccagttccat ggccaacctt agtcactact ttcggttatg gtgttcaatg ttttgcgaga
      301 tacccagatc atatgaaaca acatgacttt ttcaagtctg ccatgccaga aggttatgtt
      361 caagaaagaa ctattttttt caaagatgac ggtaactaca agaccagagc tgaagtcaag
      421 tttgaaggtg ataccttagt taatagaatc gaattaaaag gtattgattt taaagaagat
      481 ggtaacattt taggtcacaa attggaatac aactataact ctcacaatgt ttacatcatg
      541 gctgacaaac aaaagaatgg tatcaaagtt aacttcaaaa ttagacacaa cattgaagat
      601 ggttctgttc aattagctga ccattatcaa caaaatactc caattggtga tggtccagtc
      661 ttgttaccag acaaccatta cttatccact caatctgcct tatccaaaga tccaaacgaa
      721 aagagagacc acatggtctt gttagaattt gttactgctg ctggtattac ccatggtatg
      781 gatgaattgt acaaaggttc tcaccaccac caccaccact aagcgcgcca gatctgttta
      841 gcttgcctcg tccccgccgg gtcacccggc cagcgacatg gaggcccaga ataccctcct
      901 tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc
      961 catacatccc catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag
     1021 caaaaattac ggctcctcgc tgcggacctg cgagcaggga aacgctcccc tcacagacgc
     1081 gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact
     1141 gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac
     1201 atccgaacat aaacaaccat gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag
     1261 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa
     1321 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc
     1381 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
     1441 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
     1501 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag
     1561 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
     1621 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
     1681 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
     1741 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
     1801 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
     1861 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
     1921 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
     1981 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
     2041 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
     2101 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
     2161 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
     2221 agcactcgtc cgagggcaaa ggaataatct cgagtcatgt aattagttat gtcacgctta
     2281 cattcacgcc ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa
     2341 gtctaggtcc ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt
     2401 caaatttttc ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc
     2461 ttgcttgaga aggttttggg acgctcgaag gctttaacga gctcgaattc atcgatgata
     2521 tcagatccac tagtggccta tgcggccgcg gatctgccgg tctccctata gtgagtcgta
     2581 ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc
     2641 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2701 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2761 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2821 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2881 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2941 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3001 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3061 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3121 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3181 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3241 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     3301 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3361 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3421 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3481 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3541 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3601 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3661 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3721 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3781 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3841 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3901 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3961 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4021 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4081 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4141 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4201 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4261 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4321 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4381 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4441 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4501 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4561 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     4621 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     4681 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     4741 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     4801 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg
     4861 ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga
     4921 cactata
//
