LOCUS       sequence-454065-        6342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses BioID2-FRB fusion from ADH1 promoter; yeast BioID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6342)
  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 6342)
  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-BioID2-FRB-HA3
FEATURES             Location/Qualifiers
     source          1..6342
                     /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             3927..4628
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MSFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRK
                     WLSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKK
                     VSGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRIS
                     ENLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIK
                     EILSGEFSLRRS"
     CDS             4647..4925
                     /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             4932..5021
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(5070..5090)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5070..5088)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5109..5125)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5109..5125)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5122..5144)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5133..5149
                     /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(5157..5163,5164..5181,5182..5187))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5202..5223
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5340..5357)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5511..6099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5591..5610)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6270..6342,1..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 ccttcaagaa cctgatctgg ctgaaggagg
     3961 tggacagcac ccaggagaga ctgaaggagt ggaacgtgag ctacggcacc gccctggtgg
     4021 ccgacagaca gaccaagggc agaggcggcc tgggcagaaa gtggctgagc caggagggcg
     4081 gcctgtactt cagcttcctg ctgaacccca aggagttcga gaacctgctg cagctgcccc
     4141 tggtgctggg cctgagcgtg agcgaggccc tggaggagat caccgagatc cccttcagcc
     4201 tgaagtggcc caacgacgtg tacttccagg agaagaaggt gagcggcgtg ctgtgcgagc
     4261 tgagcaagga caagctgatc gtgggcatcg gcatcaacgt gaaccagaga gagatccccg
     4321 aggagatcaa ggacagagcc accaccctgt acgagatcac cggcaaggac tgggacagaa
     4381 aggaggtgct gctgaaggtg ctgaagagaa tcagcgagaa cctgaagaag ttcaaggaga
     4441 agagcttcaa ggagttcaag ggcaagatcg agagcaagat gctgtacctg ggcgaggagg
     4501 tgaagctgct gggcgagggc aagatcaccg gcaagctggt gggcctgagc gagaagggcg
     4561 gcgccctgat cctgaccgag gagggcatca aggagatcct gagcggcgag ttcagcctga
     4621 gaagaagcgg ttctggtggt gctagcatcc tctggcatga gatgtggcat gaaggcctgg
     4681 aagaggcatc tcgtttgtac tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg
     4741 agcccttgca tgctatgatg gaacggggcc cccagactct gaaggaaaca tcctttaatc
     4801 aggcctatgg tcgagattta atggaggccc aagagtggtg caggaagtac atgaaatcag
     4861 ggaatgtcaa ggacctcctc caagcctggg acctctatta tcatgtgttc cgacgaatct
     4921 caaagctcga gtacccatac gatgttcctg actatgcggg ctatccgtat gacgtcccgg
     4981 actatgcagg atcttatcca tatgacgttc cagattacgc tgcttgatct agagcggccg
     5041 ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     5101 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5161 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5221 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5281 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5341 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5401 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5461 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5521 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5581 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5641 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5701 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5761 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5821 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5881 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5941 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6001 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6061 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6121 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6181 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6241 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6301 tcagcgatct gtctatttcg ttcatccata gttgcctgac tc
//
