LOCUS       sequence-420585-        4675 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal PCR-based tagging with CUbo(K63R)-His6 in budding yeast 
            with HygR selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4675)
  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 4675)
  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-CUbo(K63R)-His6
FEATURES             Location/Qualifiers
     source          1..4675
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             85..405
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             550..567
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        623..966
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             967..1995
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             967..1995
                     /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      2003..2250
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2019..2037)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2312..2331)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2313..2331)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2418..2435)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2589..3177)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2669..2688)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3348..4139,4140..4208))
                     /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     3971..3990
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4209..4313)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4381..4399
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4437..4459)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4559..4578
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4659..4675,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4659..4675,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acgggagcgg agcaggtgct
       61 ggtgctggtg ctggagcaat tctgggagtg caggtggaaa ccatctcccc aggagacggg
      121 cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat
      181 ggaaagaaat ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag
      241 caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc
      301 aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca
      361 ccacatgcca ctctcgtctt cgatgtggag cttctaaaac tggaagggtc gaccggtatc
      421 cctccagatc aacaaagatt gatctttgcc ggtaagcagc tagaagacgg tagaacgctg
      481 tctgattaca acattcagcg agagtccacc ttacatcttg tgctaaggct aagaggtgtt
      541 actggatccc accaccacca ccaccactaa gcgcgccaga tctgtttagc ttgcctcgtc
      601 cccgccgggt cacccggcca gcgacatgga ggcccagaat accctccttg acagtcttga
      661 cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac atttagccca tacatcccca
      721 tgtataatca tttgcatcca tacattttga tggccgcacg gcgcgaagca aaaattacgg
      781 ctcctcgctg cggacctgcg agcagggaaa cgctcccctc acagacgcgt tgaattgtcc
      841 ccacgccgcg cccctgtaga gaaatataaa aggttaggat ttgccactga ggttcttctt
      901 tcatatactt ccttttaaaa tcttgctagg atacagttct cacatcacat ccgaacataa
      961 acaaccatgg gtaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     1021 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     1081 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     1141 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     1201 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     1261 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     1321 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     1381 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     1441 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     1501 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     1561 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     1621 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     1681 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     1741 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     1801 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     1861 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     1921 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     1981 agggcaaagg aataatctcg agtcatgtaa ttagttatgt cacgcttaca ttcacgccct
     2041 ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct
     2101 atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt
     2161 ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag
     2221 gttttgggac gctcgaaggc tttaacgagc tcgaattcat cgatgatatc agatccacta
     2281 gtggcctatg cggccgcgga tctgccggtc tccctatagt gagtcgtatt aatttcgata
     2341 agccaggtta acctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2401 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2461 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2521 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2581 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2641 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2701 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2761 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2821 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2881 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2941 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3001 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3061 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3121 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3181 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3241 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3301 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3361 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3421 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3481 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3541 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3601 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3661 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3721 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3781 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3841 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3901 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3961 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4021 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4081 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4141 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4201 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4261 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4321 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     4381 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     4441 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     4501 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     4561 gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc
     4621 tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca ctata
//
