LOCUS       sequence-454144-        5444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For TurboID-FRB integration; yeast BioID with HIS3MX6 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5444)
  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 5444)
  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-HIS3MX6
FEATURES             Location/Qualifiers
     source          1..5444
                     /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"
     promoter        1732..2075
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2076..2774
                     /codon_start=1
                     /product="S. kluyveri imidazoleglycerol phosphate 
                     dehydratase"
                     /label=k-HIS3
                     /translation="MAEPAQKKQKQTVQERKAFISRITNETKIQIAISLNGGYIQIKDS
                     ILPAKKDDDVASQATQSQVIDIHTGVGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHT
                     TEDCGIALGQAFKEAMGAVRGVKRFGTGFAPLDEALSRAVVDLSSRPFAVIDLGLKREM
                     IGDLSTEMIPHFLESFAEAARITLHVDCLRGFNDHHRSESAFKALAVAIREAISSNGTN
                     DVPSTKGVLM"
     terminator      2778..2975
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3079..3098)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3080..3098)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3185..3202)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3356..3944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3436..3455)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4115..4906,4907..4975))
                     /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     4738..4757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4976..5080)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5148..5166
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5204..5226)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5326..5345
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5426..5444
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5426..5443
                     /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 caaccatggc agaaccagcc caaaaaaagc
     2101 aaaaacaaac tgttcaggag cgcaaggcgt ttatctcccg tatcactaat gaaactaaaa
     2161 ttcaaatcgc tatttcgctg aatggtggtt atattcaaat aaaagattcg attcttcctg
     2221 caaagaagga tgacgatgta gcttcccaag ctactcagtc acaggtcatc gatattcaca
     2281 caggtgttgg ctttttggat catatgatcc atgcgttggc aaaacactct ggttggtctc
     2341 ttattgttga atgtattggt gacctgcaca ttgacgatca ccatactacc gaagattgcg
     2401 gtatcgcatt agggcaagcg ttcaaagaag caatgggtgc tgtccgtggt gtaaaaagat
     2461 tcggtactgg gttcgcacca ttggatgagg cgctatcacg tgccgtagtc gatttatcta
     2521 gtagaccatt tgctgtaatc gaccttggat tgaagagaga gatgattggt gatttatcca
     2581 ctgaaatgat tccacacttt ttggaaagtt tcgcggaggc ggccagaatt actttgcatg
     2641 ttgattgtct gagaggtttc aacgatcacc acagaagtga gagtgcgttc aaggctttgg
     2701 ctgttgccat aagagaagct atttctagca atggcaccaa tgacgttccc tcaaccaaag
     2761 gtgttttgat gtgaagtact gacaataaaa agattcttgt tttcaagaac ttgtcatttg
     2821 tatagttttt ttatattgta gttgttctat tttaatcaaa tgttagcgtg atttatattt
     2881 tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat
     2941 gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc gattcgatac taacgccgcc
     3001 atccagttta aacgagctcg aattcatcga tgatatcaga tccactagtg gcctatgcgg
     3061 ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc caggttaacc
     3121 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3181 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3241 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3301 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3361 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3421 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3481 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3541 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3601 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3661 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3721 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3781 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3841 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3901 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3961 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4021 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4081 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4141 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4201 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4261 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4321 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4381 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4441 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4501 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4561 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4621 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4681 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4741 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4801 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4861 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4921 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4981 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5041 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5101 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     5161 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     5221 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     5281 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     5341 ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca
     5401 taaccttatg tatcatacac atacgattta ggtgacacta taga
//
