LOCUS       sequence-454145-        5557 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For TurboID-FRB integration; yeast BioID with KanMX6 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5557)
  AUTHORS   Pfannenstein J, Tyryshkin M, Gulden ME, Doud EH, Mosley AL, Reese JC
  TITLE     Characterization of BioID tagging systems in budding yeast and 
            exploring the interactome of the Ccr4-Not complex.
  JOURNAL   G3 (Bethesda). 2024 Sep 13:jkae221. doi: 10.1093/g3journal/jkae221.
  PUBMED    39271111
REFERENCE   2  (bases 1 to 5557)
  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: "G3 
            (Bethesda). 2024 Sep 13:jkae221. doi: 10.1093/g3journal/jkae221."
COMMENT     Sequence Label: pFa6-TID-FRB-3HA-KanMX6
FEATURES             Location/Qualifiers
     source          1..5557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1069..1347
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             1354..1443
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      1470..1657
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1525..1549)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1732..3088
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1732..2075
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2076..2885
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2143..2162)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2891..3088
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3192..3211)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3193..3211)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3298..3315)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3469..4057)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3549..3568)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4228..5019,5020..5088))
                     /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     4851..4870
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5089..5193)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5261..5279
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5317..5339)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5439..5458
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5539..5557
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5539..5556
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 acgcggccgc cagctgaagc ttcgtacgct gcaggtcgac ggatccccgg gttaattaac
       61 gcaagagatc cgcctgtggc cacgatgaaa gataacacgg ttcccttaaa attgatagcc
      121 cttcttgcca atggtgagtt tcattccggg gaacaacttg gcgaaactct gggaatgagc
      181 agggcagcga ttaacaagca tatccaaacc ttgagagact ggggagtaga tgtgttcacc
      241 gtgcctggta aaggttattc tttgccggaa cctatccctc tattaaatgc gaaacagata
      301 ctaggacaac tggacggtgg tagtgttgcc gtattgccgg ttgttgactc aaccaatcaa
      361 tacttgctgg acagaattgg cgaacttaaa agcggtgatg cgtgtattgc agaatatcag
      421 caagcaggga gaggctcaag aggtaggaaa tggttttcac cttttggcgc caatctttac
      481 ttaagtatgt tctggcgttt gaagagaggg cccgcggcta ttggtttagg tcccgttatc
      541 gggatagtta tggctgaagc gctacgtaaa cttggggccg acaaggtcag agtgaagtgg
      601 cctaatgatc tatacttgca agatagaaag ctagcgggta tcctggtgga gctagcggga
      661 atcacagggg acgctgccca aatagtaatc ggtgctggta taaatgtcgc aatgcgtaga
      721 gtcgaggaat cagtggtgaa ccagggctgg atcacgttgc aagaggcggg cataaacctg
      781 gaccgtaaca cccttgcagc gaccctaata agagaactac gtgctgcatt ggagcttttt
      841 gagcaggagg gcctggctcc gtacctaccc aggtgggaga aattagacaa tttcataaac
      901 cgtccagtca aactaataat tggagataag gagattttcg gtatttctcg tgggatagac
      961 aagcagggag cgttgttatt agaacaggat ggtgtcatta aaccttggat gggcggtgaa
     1021 attagtcttc gtagtgccga aaaggctagc ggttctggtg gtgctagcat cctctggcat
     1081 gagatgtggc atgaaggcct ggaagaggca tctcgtttgt actttgggga aaggaacgtg
     1141 aaaggcatgt ttgaggtgct ggagcccttg catgctatga tggaacgggg cccccagact
     1201 ctgaaggaaa catcctttaa tcaggcctat ggtcgagatt taatggaggc ccaagagtgg
     1261 tgcaggaagt acatgaaatc agggaatgtc aaggacctcc tccaagcctg ggacctctat
     1321 tatcatgtgt tccgacgaat ctcaaagctc gagtacccat acgatgttcc tgactatgcg
     1381 ggctatccgt atgacgtccc ggactatgca ggatcttatc catatgacgt tccagattac
     1441 gctgcttgag gcgcgccact tctaaataag cgaatttctt atgatttatg atttttatta
     1501 ttaaataagt tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa
     1561 aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat
     1621 agtatgaggt cgctcttatt gaccacacct ctaccggcag atccgctagg gataacaggg
     1681 taatatagat ctgtttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag
     1741 gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt
     1801 cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat acattttgat
     1861 ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc agacctgcga gcagggaaac
     1921 gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa
     1981 ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga
     2041 tacagttctc acatcacatc cgaacataaa caaccatggg taaggaaaag actcacgttt
     2101 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     2161 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag
     2221 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     2281 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     2341 ctgatgatgc atggttactc accactgcga tccccggcaa aacagcattc caggtattag
     2401 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     2461 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     2521 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     2581 atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg
     2641 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     2701 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     2761 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     2821 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     2881 tctaatcagt actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt
     2941 tttttatatt gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg
     3001 cctcgacatc atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa
     3061 tcgtatgtga atgctggtcg ctatactgct gtcgattcga tactaacgcc gccatccagt
     3121 ttaaacgagc tcgaattcat cgatgatatc agatccacta gtggcctatg cggccgcgga
     3181 tctgccggtc tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta
     3241 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3301 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3361 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3421 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3481 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3541 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3601 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3661 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3721 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3781 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3841 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3901 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3961 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4021 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4081 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4141 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4201 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4261 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4321 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4381 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4441 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4501 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4561 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4621 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4681 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4741 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4801 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4861 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4921 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4981 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5041 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5101 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5161 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5221 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     5281 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     5341 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     5401 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     5461 gagagtgcac catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt
     5521 atgtatcata cacatacgat ttaggtgaca ctataga
//
