LOCUS       sequence-451885-        5580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To integrate TID-FRB-NLS fusion at yeast promoter; yeast BioID with 
            KanMX6 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5580)
  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 5580)
  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-KanMX6
FEATURES             Location/Qualifiers
     source          1..5580
                     /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      1493..1680
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1548..1572)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1755..3111
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1755..2098
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2099..2908
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2166..2185)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2914..3111
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3215..3234)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3216..3234)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3321..3338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3492..4080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3572..3591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4251..5042,5043..5111))
                     /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     4874..4893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5112..5216)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5284..5302
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5340..5362)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5462..5481
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5562..5580
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5562..5579
                     /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 gaggcgcgcc acttctaaat aagcgaattt
     1501 cttatgattt atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt
     1561 ttaaagtgac tcttaggttt taaaacgaaa attcttattc ttgagtaact ctttcctgta
     1621 ggtcaggttg ctttctcagg tatagtatga ggtcgctctt attgaccaca cctctaccgg
     1681 cagatccgct agggataaca gggtaatata gatctgttta gcttgcctcg tccccgccgg
     1741 gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca
     1801 gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat
     1861 catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc
     1921 tgcagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg
     1981 cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac
     2041 ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat
     2101 gggtaaggaa aagactcacg tttcgaggcc gcgattaaat tccaacatgg atgctgattt
     2161 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt
     2221 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     2281 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     2341 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     2401 caaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     2461 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     2521 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     2581 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     2641 taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     2701 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     2761 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     2821 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     2881 tcatttgatg ctcgatgagt ttttctaatc agtactgaca ataaaaagat tcttgttttc
     2941 aagaacttgt catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt
     3001 agcgtgattt atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg
     3061 cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt
     3121 cgatactaac gccgccatcc agtttaaacg agctcgaatt catcgatgat atcagatcca
     3181 ctagtggcct atgcggccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg
     3241 ataagccagg ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3301 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3361 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3421 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3481 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3541 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3601 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3661 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3721 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3781 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3841 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3901 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3961 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4021 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4081 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4141 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4201 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4261 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4321 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4381 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4441 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4501 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4561 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4621 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4681 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4741 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4801 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4861 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4921 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4981 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5041 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5101 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5161 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5221 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     5281 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     5341 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     5401 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     5461 gcggcatcag agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc
     5521 ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg acactataga
//
