LOCUS       sequence-453556-        6916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TurboID-FRB-NLS fusion from CUP1 promoter; yeast BioID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6916)
  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 6916)
  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-CUP1p-TID-FRB-NLS-HA3
FEATURES             Location/Qualifiers
     source          1..6916
                     /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        3890..4124
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             5197..5475
                     /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             5485..5505
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5506..5595
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(5644..5664)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5644..5662)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5683..5699)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5683..5699)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5696..5718)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5707..5723
                     /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(5731..5737,5738..5755,5756..5761))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5776..5797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5914..5931)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6085..6673)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6165..6184)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6844..6916,1..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 ttgggtacca gattatagtc agtcggcgaa gaacaaaaat gttctcttga
     3541 aattccccgt tagtgaactg aacagaatct atttaaaatt taaggagatt tcagattttt
     3601 taatggaaag agaagttgtc caaaggagta taattattga caaggatttg gaatctgata
     3661 atctgggtat tactacggca aacttcaacg atttctatga tgcattttat aattagtaag
     3721 ccgatcccat taccgacatt tgggcgctat acgtgcatat gttcatgtat gtatctgtat
     3781 ttaaaacact tttgtattat ttttcctcat atatgtgtat aggtttatac ggatgattta
     3841 attattactt caccaccctt tatttcaggc tgatatctta gccttgttac tagttagaaa
     3901 aagacatttt tgctgtcagt cactgtcaag agattctttt gctggcattt cttctagaag
     3961 caaaaagagc gatgcgtctt ttccgctgaa ccgttccagc aaaaaagact accaacgcaa
     4021 tatggattgt cagaatcata taaaagagaa gcaaataact ccttgtcttg tatcaattgc
     4081 attataatat cttcttgtta gtgcaatatc atatagaagt catcgaaata gatattaaga
     4141 aaaacaaact gtacaatcaa tcaatcaatc atcacaaagc ttaaaatggc aagagatccg
     4201 cctgtggcca cgatgaaaga taacacggtt cccttaaaat tgatagccct tcttgccaat
     4261 ggtgagtttc attccgggga acaacttggc gaaactctgg gaatgagcag ggcagcgatt
     4321 aacaagcata tccaaacctt gagagactgg ggagtagatg tgttcaccgt gcctggtaaa
     4381 ggttattctt tgccggaacc tatccctcta ttaaatgcga aacagatact aggacaactg
     4441 gacggtggta gtgttgccgt attgccggtt gttgactcaa ccaatcaata cttgctggac
     4501 agaattggcg aacttaaaag cggtgatgcg tgtattgcag aatatcagca agcagggaga
     4561 ggctcaagag gtaggaaatg gttttcacct tttggcgcca atctttactt aagtatgttc
     4621 tggcgtttga agagagggcc cgcggctatt ggtttaggtc ccgttatcgg gatagttatg
     4681 gctgaagcgc tacgtaaact tggggccgac aaggtcagag tgaagtggcc taatgatcta
     4741 tacttgcaag atagaaagct agcgggtatc ctggtggagc tagcgggaat cacaggggac
     4801 gctgcccaaa tagtaatcgg tgctggtata aatgtcgcaa tgcgtagagt cgaggaatca
     4861 gtggtgaacc agggctggat cacgttgcaa gaggcgggca taaacctgga ccgtaacacc
     4921 cttgcagcga ccctaataag agaactacgt gctgcattgg agctttttga gcaggagggc
     4981 ctggctccgt acctacccag gtgggagaaa ttagacaatt tcataaaccg tccagtcaaa
     5041 ctaataattg gagataagga gattttcggt atttctcgtg ggatagacaa gcagggagcg
     5101 ttgttattag aacaggatgg tgtcattaaa ccttggatgg gcggtgaaat tagtcttcgt
     5161 agtgccgaaa aggctagcgg ttctggtggt gctagcatcc tctggcatga gatgtggcat
     5221 gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa ggaacgtgaa aggcatgttt
     5281 gaggtgctgg agcccttgca tgctatgatg gaacggggcc cccagactct gaaggaaaca
     5341 tcctttaatc aggcctatgg tcgagattta atggaggccc aagagtggtg caggaagtac
     5401 atgaaatcag ggaatgtcaa ggacctcctc caagcctggg acctctatta tcatgtgttc
     5461 cgacgaatct caaagctcga gccaccaaaa aagaaaagaa aagtttaccc atacgatgtt
     5521 cctgactatg cgggctatcc gtatgacgtc ccggactatg caggatctta tccatatgac
     5581 gttccagatt acgctgcttg atctagagcg gccgccaccg cggtggagct ccagcttttg
     5641 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     5701 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5761 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5821 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5881 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5941 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6001 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6061 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6121 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6181 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6241 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6301 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6361 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6421 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6481 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6541 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6601 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6661 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6721 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6781 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6841 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6901 catagttgcc tgactc
//
