LOCUS       sequence-451857-        5275 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To insert biotin ligase, TurboID, at the C-terminus of genes; yeast 
            BioID, KanMX6 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5275)
  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 5275)
  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: pFA6a-TID-HA3-KanMX6
FEATURES             Location/Qualifiers
     source          1..5275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1066..1155
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      1188..1375
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1243..1267)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1450..2806
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1450..1793
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1794..2603
                     /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(1861..1880)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2609..2806
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2910..2929)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2911..2929)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3016..3033)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3187..3775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3267..3286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3946..4737,4738..4806))
                     /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     4569..4588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4807..4911)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4979..4997
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5035..5057)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5157..5176
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5257..5275
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5257..5274
                     /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 ttaattaaca tcttttaccc atacgatgtt
     1081 cctgactatg cgggctatcc ctatgacgtc ccggactatg caggatccta tccatatgac
     1141 gttccagatt acgctgctca gtgctgaggc gcgccacttc taaataagcg aatttcttat
     1201 gatttatgat ttttattatt aaataagtta taaaaaaaat aagtgtatac aaattttaaa
     1261 gtgactctta ggttttaaaa cgaaaattct tattcttgag taactctttc ctgtaggtca
     1321 ggttgctttc tcaggtatag tatgaggtcg ctcttattga ccacacctct accggcagat
     1381 ccgctaggga taacagggta atatagatct gtttagcttg cctcgtcccc gccgggtcac
     1441 ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca
     1501 ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt
     1561 gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcag
     1621 acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc
     1681 ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct
     1741 tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca accatgggta
     1801 aggaaaagac tcacgtttcg aggccgcgat taaattccaa catggatgct gatttatatg
     1861 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
     1921 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     1981 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     2041 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggcaaaa
     2101 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     2161 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     2221 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     2281 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc
     2341 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     2401 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     2461 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     2521 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     2581 tgatgctcga tgagtttttc taatcagtac tgacaataaa aagattcttg ttttcaagaa
     2641 cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt
     2701 gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa
     2761 agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt cgattcgata
     2821 ctaacgccgc catccagttt aaacgagctc gaattcatcg atgatatcag atccactagt
     2881 ggcctatgcg gccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag
     2941 ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3001 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3061 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3121 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3181 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3241 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3301 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3361 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3421 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3481 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3541 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3601 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3661 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3721 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3781 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3841 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3901 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3961 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4021 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4081 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4141 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4201 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4261 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4321 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4381 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4441 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4501 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4561 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4621 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4681 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4741 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4801 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4861 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4921 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     4981 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     5041 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     5101 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     5161 atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta
     5221 caattaatac ataaccttat gtatcataca catacgattt aggtgacact ataga
//
