LOCUS       sequence-420588-        4174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal PCR-based tagging with His6 in budding yeast with HygR 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4174)
  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 4174)
  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-His6
FEATURES             Location/Qualifiers
     source          1..4174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             49..66
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        122..465
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             466..1494
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             466..1494
                     /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      1502..1749
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1518..1536)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1811..1830)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1812..1830)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1917..1934)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2088..2676)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2168..2187)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2847..3638,3639..3707))
                     /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     3470..3489
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3708..3812)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3880..3898
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3936..3958)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4058..4077
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4158..4174,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4158..4174,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatccca ccaccaccac
       61 caccactaag cgcgccagat ctgtttagct tgcctcgtcc ccgccgggtc acccggccag
      121 cgacatggag gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg
      181 atgtgactgt cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat
      241 acattttgat ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc ggacctgcga
      301 gcagggaaac gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag
      361 aaatataaaa ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat
      421 cttgctagga tacagttctc acatcacatc cgaacataaa caaccatggg taaaaagcct
      481 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
      541 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
      601 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
      661 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
      721 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
      781 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
      841 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
      901 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
      961 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     1021 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     1081 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     1141 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     1201 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     1261 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     1321 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     1381 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     1441 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga ataatctcga
     1501 gtcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc
     1561 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg
     1621 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg
     1681 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct
     1741 ttaacgagct cgaattcatc gatgatatca gatccactag tggcctatgc ggccgcggat
     1801 ctgccggtct ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa
     1861 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     1921 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1981 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2041 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2101 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2161 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2221 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2281 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2341 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2401 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2461 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2521 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2581 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2641 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2701 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2761 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2821 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2881 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2941 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3001 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3061 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3121 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3181 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3241 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3301 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3361 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3421 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3481 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3541 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3601 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3661 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3721 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3781 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3841 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     3901 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     3961 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     4021 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     4081 agagtgcacc atatggacat attgtcgtta gaacgcggct acaattaata cataacctta
     4141 tgtatcatac acatacgatt taggtgacac tata
//
