LOCUS       sequence-255832-        4862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template for genomic C-terminal Cub-R-URA3 tag, HISMX 
            complements the histidine auxotrophy of S. cerevisiae his mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4862)
  AUTHORS   Dunkler A, Muller J, Johnsson N
  TITLE     Detecting protein-protein interactions with the Split-Ubiquitin 
            sensor.
  JOURNAL   Methods Mol Biol. 2012;786:115-30. doi: 10.1007/978-1-61779-292-2_7.
  PUBMED    21938623
REFERENCE   2  (bases 1 to 4862)
  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: "Methods Mol
            Biol."; date: "2012"; volume: "786:115-30. doi"; pages: " 
            10.1007/978-1-61779-292-2_7"
COMMENT     Sequence Label: pFA CRU HISMX
FEATURES             Location/Qualifiers
     source          1..4862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             307..333
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             349..1149
                     /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="SKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELLE
                     LVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQY
                     SAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGEY
                     TKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDDV
                     VSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     gene            1195..2395
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        1195..1538
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1539..2192
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S. 
                     cerevisiae HIS3"
                     /translation="MGRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHANQ
                     KGEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQ
                     AMGNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLY
                     SFSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      2198..2395
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2499..2518)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2500..2518)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2605..2622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2776..3364)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2856..2875)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3535..4326,4327..4395))
                     /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     4158..4177
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4396..4500)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4568..4586
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4624..4646)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4746..4765
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4846..4862,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4846..4862,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggtatccc tccagatcaa
       61 caaagattga tctttgccgg taagcagcta gaagacggta gaacgctgtc tgattacaac
      121 attcagaagg agtccacctt acatcttgtg ctaaggctaa gaggtggtag gcacggatcc
      181 ggagcttggc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
      241 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
      301 cgtggctacc catacgacgt cccagattac gccgctttcc ttggccagtc gaaagctaca
      361 tataaggaac gtgctgctac tcatcctagt cctgttgctg ccaagctatt taatatcatg
      421 cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa ggaattactg
      481 gagttagttg aagcattagg tcccaaaatt tgtttactaa aaacacatgt ggatatcttg
      541 actgattttt ccatggaggg cacagttaag ccgctaaagg cattatccgc caagtacaat
      601 tttttactct tcgaagacag aaaatttgct gacattggta atacagtcaa attgcagtac
      661 tctgcgggtg tatacagaat agcagaatgg gcagacatta cgaatgcaca cggtgtggtg
      721 ggcccaggta ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa ggaacctaga
      781 ggccttttga tgttagcaga attgtcatgc aagggctccc tatctactgg agaatatact
      841 aagggtactg ttgacattgc gaagagcgac aaagattttg ttatcggctt tattgctcaa
      901 agagacatgg gtggaagaga tgaaggttac gattggttga ttatgacacc cggtgtgggt
      961 ttagatgaca agggagacgc attgggtcaa cagtatagaa ccgtggatga tgtggtctct
     1021 acaggatctg acattattat tgttggaaga ggactatttg caaagggaag ggatgctaag
     1081 gtagagggtg aacgttacag aaaagcaggc tgggaagcat atttgagaag atgcggccag
     1141 caaaactaaa gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg
     1201 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac
     1261 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt
     1321 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga
     1381 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata
     1441 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta
     1501 ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaggagg gcttttgtag
     1561 aaagaaatac gaacgaaacg aaaatcagcg ttgccatcgc tttggacaaa gctcccttac
     1621 ctgaagagtc gaattttatt gatgaactta taacttccaa gcatgcaaac caaaagggag
     1681 aacaagtaat ccaagtagac acgggaattg gattcctgga tcacatgtat catgcactgg
     1741 ctaaacatgc aggctggagc ttacgacttt actcaagagg tgatttaatc atcgatgatc
     1801 atcacactgc agaagatact gctattgcac ttggtattgc attcaagcag gctatgggta
     1861 actttgccgg cgttaaaaga tttggacatg cttattgtcc acttgacgaa gccctttcta
     1921 gaagcgtagt tgacttgtcg ggacggccct atgctgttat cgatttggga ttaaagcgtg
     1981 aaaaggttgg ggaattgtcc tgtgaaatga tccctcactt actatattcc ttttcggtag
     2041 cagctggaat tactttgcat gttacctgct tatatggtag taatgaccat catcgtgctg
     2101 aaagcgcttt taaatctctg gctgttgcca tgcgcgcggc tactagtctt actggaagtt
     2161 ctgaagtccc aagcacgaag ggagtgttgt aaagagtact gacaataaaa agattcttgt
     2221 tttcaagaac ttgtcatttg tatagttttt ttatattgta gttgttctat tttaatcaaa
     2281 tgttagcgtg atttatattt tttttcgcct cgacatcatc tgcccagatg cgaagttaag
     2341 tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc
     2401 gattcgatac taacgccgcc atccagttta aacgagctcg aattcatcga tgatatcaga
     2461 tccactagtg gcctatgcgg ccgcggatct gccggtctcc ctatagtgag tcgtattaat
     2521 ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     2581 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     2641 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     2701 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2761 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2821 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2881 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2941 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3001 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3061 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3121 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3181 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3241 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3301 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3361 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3421 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3481 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3541 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3601 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3661 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3721 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3781 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3841 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3901 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3961 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4021 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4081 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4141 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4201 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4261 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4321 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4381 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4441 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4501 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     4561 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     4621 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     4681 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     4741 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga
     4801 acgcggctac aattaataca taaccttatg tatcatacac atacgattta ggtgacacta
     4861 ta
//
