LOCUS       sequence-454143-        5468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To integrate TID-FRB-NLS fusion at yeast promoter; yeast BioID with 
            HIS3MX6 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5468)
  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 5468)
  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-NLS-3HA-HIS3MX6
FEATURES             Location/Qualifiers
     source          1..5468
                     /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             1357..1377
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1378..1467
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      1494..1681
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1549..1573)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        1756..2099
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2100..2798
                     /codon_start=1
                     /product="S. kluyveri imidazoleglycerol phosphate 
                     dehydratase"
                     /label=k-HIS3
                     /translation="MAEPAQKKQKQTVQERKAFISRITNETKIQIAISLNGGYIQIKDS
                     ILPAKKDDDVASQATQSQVIDIHTGVGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHT
                     TEDCGIALGQAFKEAMGAVRGVKRFGTGFAPLDEALSRAVVDLSSRPFAVIDLGLKREM
                     IGDLSTEMIPHFLESFAEAARITLHVDCLRGFNDHHRSESAFKALAVAIREAISSNGTN
                     DVPSTKGVLM"
     terminator      2802..2999
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3103..3122)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3104..3122)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3209..3226)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3380..3968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3460..3479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4139..4930,4931..4999))
                     /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     4762..4781
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5000..5104)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5172..5190
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5228..5250)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5350..5369
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5450..5468
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5450..5467
                     /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 gagccaccaa aaaagaaaag aaaagtttac
     1381 ccatacgatg ttcctgacta tgcgggctat ccgtatgacg tcccggacta tgcaggatct
     1441 tatccatatg acgttccaga ttacgctgct tgaggcgcgc cacttctaaa taagcgaatt
     1501 tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat
     1561 tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt
     1621 aggtcaggtt gctttctcag gtatagtatg aggtcgctct tattgaccac acctctaccg
     1681 gcagatccgc tagggataac agggtaatat agatctgttt agcttgcctc gtccccgccg
     1741 ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct tgacgtgcgc
     1801 agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc ccatgtataa
     1861 tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg
     1921 ctgcagacct gcgagcaggg aaacgctccc ctcacagacg cgttgaattg tccccacgcc
     1981 gcgcccctgt agagaaatat aaaaggttag gatttgccac tgaggttctt ctttcatata
     2041 cttcctttta aaatcttgct aggatacagt tctcacatca catccgaaca taaacaacca
     2101 tggcagaacc agcccaaaaa aagcaaaaac aaactgttca ggagcgcaag gcgtttatct
     2161 cccgtatcac taatgaaact aaaattcaaa tcgctatttc gctgaatggt ggttatattc
     2221 aaataaaaga ttcgattctt cctgcaaaga aggatgacga tgtagcttcc caagctactc
     2281 agtcacaggt catcgatatt cacacaggtg ttggcttttt ggatcatatg atccatgcgt
     2341 tggcaaaaca ctctggttgg tctcttattg ttgaatgtat tggtgacctg cacattgacg
     2401 atcaccatac taccgaagat tgcggtatcg cattagggca agcgttcaaa gaagcaatgg
     2461 gtgctgtccg tggtgtaaaa agattcggta ctgggttcgc accattggat gaggcgctat
     2521 cacgtgccgt agtcgattta tctagtagac catttgctgt aatcgacctt ggattgaaga
     2581 gagagatgat tggtgattta tccactgaaa tgattccaca ctttttggaa agtttcgcgg
     2641 aggcggccag aattactttg catgttgatt gtctgagagg tttcaacgat caccacagaa
     2701 gtgagagtgc gttcaaggct ttggctgttg ccataagaga agctatttct agcaatggca
     2761 ccaatgacgt tccctcaacc aaaggtgttt tgatgtgaag tactgacaat aaaaagattc
     2821 ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat
     2881 caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt
     2941 taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgc
     3001 tgtcgattcg atactaacgc cgccatccag tttaaacgag ctcgaattca tcgatgatat
     3061 cagatccact agtggcctat gcggccgcgg atctgccggt ctccctatag tgagtcgtat
     3121 taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3181 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3241 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3301 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3361 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3421 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3481 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3541 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3601 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3661 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3721 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3781 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3841 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3901 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3961 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4021 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4081 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4141 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4201 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4261 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4321 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4381 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4441 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4501 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4561 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4621 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4681 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4741 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4801 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4861 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4921 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4981 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5041 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5101 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     5161 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     5221 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     5281 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     5341 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt
     5401 tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac
     5461 actataga
//
