LOCUS       sequence-454072-        6630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses BirA-FRB-NLS fusion from ADH1 promoter; yeast BioID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6630)
  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 6630)
  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: pRS416-ADH1p-BirA-FRB-NLS-HA3
FEATURES             Location/Qualifiers
     source          1..6630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     551..570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(789..893)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    930..1433
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     1474..1492
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1530..1552)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1652..1671
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1695..1910
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1911..2714
                     /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="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(2845..3300)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2982..3003)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3194..3213
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3426..3448
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3440..3457
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3441..3457
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3467..3486
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3467..3485
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3495..3896
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     primer_bind     3901..3917
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3902..3918
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(3927..4280,4281..4283,4284..4892)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="MSKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLR
                     DWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKS
                     GDACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLR
                     KLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQ
                     GWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLII
                     GDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             4911..5189
                     /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             5199..5219
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5220..5309
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(5358..5378)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5358..5376)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5397..5413)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5397..5413)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5410..5432)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5421..5437
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5445..5451,5452..5469,5470..5475))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5490..5511
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5628..5645)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5799..6387)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5879..5898)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6558..6630,1..719,720..788))
                     /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"
ORIGIN
        1 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
       61 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
      121 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
      181 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
      241 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
      301 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
      361 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
      421 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
      481 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
      541 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      601 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      661 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      721 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      781 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      841 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      901 ccccgaaaag tgccacctgg gtccttttca tcacgtgcta taaaaataat tataatttaa
      961 attttttaat ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa
     1021 atagtttttg ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct
     1081 tttatcttgc tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta
     1141 gaagaccaca cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata
     1201 tctatttaat ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa
     1261 attttttaaa cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat
     1321 ttactttcta aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa
     1381 attattccat cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtctaa
     1441 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     1501 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     1561 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     1621 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     1681 caccatacca ccttttcaat tcatcatttt ttttttattc ttttttttga tttcggtttc
     1741 cttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca
     1801 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat
     1861 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag
     1921 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata
     1981 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat
     2041 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata
     2101 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt
     2161 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc
     2221 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg
     2281 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac
     2341 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat
     2401 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg
     2461 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg
     2521 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg
     2581 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg
     2641 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg
     2701 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta
     2761 gagcttcaat ttaattatat cagttattac cctatgcggt gtgaaatacc gcacagatgc
     2821 gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta aaattcgcgt
     2881 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     2941 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3001 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3061 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3121 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3181 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3241 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3301 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     3361 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     3421 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     3481 atagggcgaa ttgggtaccc aacttctttt cttttttttt cttttctctc tcccccgttg
     3541 ttgtctcacc atatccgcaa tgacaaaaaa atgatggaag acactaaagg aaaaaattaa
     3601 cgacaaagac agcaccaaca gatgtcgttg ttccagagct gatgaggggt atctcgaagc
     3661 acacgaaact ttttccttcc ttcattcacg cacactactc tctaatgagc aacggtatac
     3721 ggccttcctt ccagttactt gaatttgaaa taaaaaaaag tttgctgtct tgctatcaag
     3781 tataaataga cctgcaatta ttaatctttt gtttcctcgt cattgttctc gttccctttc
     3841 ttccttgttt ctttttctgc acaatatttc aagctatacc aagcatacaa tcaactccac
     3901 tcgaggtcga cggtatcgat aagcttatgt ccaaggacaa caccgtgccc ctgaagctga
     3961 tcgccctgct ggccaacggc gagttccact ctggcgagca gctgggagag accctgggaa
     4021 tgagcagagc cgccatcaac aagcacatcc agacactgag agactgggga gtggacgtgt
     4081 tcaccgtgcc tggcaagggc tacagcctgc ctgagcctat ccagctgctg aacgccaagc
     4141 agatcctggg acagctggat ggcggaagcg tggccgtgct gcctgtgatc gactccacca
     4201 atcagtacct gctggacaga atcggagagc tgaagtccgg cgacgcctgc atcgccgagt
     4261 accagcaggc tggcagagga ggcagaggac ggaagtggtt cagcccattc ggagccaacc
     4321 tgtacctgtc catgttctgg agactggagc agggacctgc tgctgccatc ggactgagtc
     4381 tggtgatcgg aatcgtgatg gccgaggtgc tgagaaagct gggagccgac aaggtgagag
     4441 tgaagtggcc taatgacctg tacctccagg accgcaagct ggctggcatc ctggtggagc
     4501 tgacaggcaa gacaggcgat gccgctcaga tcgtgatcgg agccggaatc aacatggcca
     4561 tgagaagagt ggaggagagc gtggtgaacc agggctggat caccctgcag gaggctggca
     4621 tcaacctgga ccggaacacc ctggccgcca tgctgatcag agagctgaga gccgctctgg
     4681 agctgttcga gcaggaggga ctggctcctt acctgagcag atgggagaag ctggacaact
     4741 tcatcaacag acctgtgaag ctgatcatcg gcgacaagga aatcttcggc atctccagag
     4801 gaatcgacaa gcagggagct ctgctgctgg agcaggacgg aatcatcaag ccctggatgg
     4861 gcggagaaat ctccctgaga agcgcagaga agggttctgg tggtgctagc atcctctggc
     4921 atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg
     4981 tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga
     5041 ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt
     5101 ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cctccaagcc tgggacctct
     5161 attatcatgt gttccgacga atctcaaagc tcgagccacc aaaaaagaaa agaaaagttt
     5221 acccatacga tgttcctgac tatgcgggct atccgtatga cgtcccggac tatgcaggat
     5281 cttatccata tgacgttcca gattacgctg cttgatctag agcggccgcc accgcggtgg
     5341 agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     5401 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5461 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5521 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5581 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5641 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5701 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5761 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5821 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5881 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5941 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6001 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6061 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6121 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6181 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6241 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6301 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6361 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6421 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6481 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6541 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6601 ctatttcgtt catccatagt tgcctgactc
//
