LOCUS       sequence-451855-        4981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To insert HA-tagged biotin ligase, BioID2, 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 4981)
  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 4981)
  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-BioID2-HA3-KanMX6
FEATURES             Location/Qualifiers
     source          1..4981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             61..756
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="FKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKWL
                     SQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKVS
                     GVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISEN
                     LKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKEI
                     LSGEFSLRRS"
     CDS             772..861
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      894..1081
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(949..973)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1156..2512
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1156..1499
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1500..2309
                     /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(1567..1586)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2315..2512
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2616..2635)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2617..2635)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2722..2739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2893..3481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2973..2992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3652..4443,4444..4512))
                     /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     4275..4294
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4513..4617)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4685..4703
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4741..4763)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4863..4882
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4963..4981
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4963..4980
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 acgcggccgc cagctgaagc ttcgtacgct gcaggtcgac ggatccccgg gttaattaac
       61 ttcaagaacc tgatctggct gaaggaggtg gacagcaccc aggagagact gaaggagtgg
      121 aacgtgagct acggcaccgc cctggtggcc gacagacaga ccaagggcag aggcggcctg
      181 ggcagaaagt ggctgagcca ggagggcggc ctgtacttca gcttcctgct gaaccccaag
      241 gagttcgaga acctgctgca gctgcccctg gtgctgggcc tgagcgtgag cgaggccctg
      301 gaggagatca ccgagatccc cttcagcctg aagtggccca acgacgtgta cttccaggag
      361 aagaaggtga gcggcgtgct gtgcgagctg agcaaggaca agctgatcgt gggcatcggc
      421 atcaacgtga accagagaga gatccccgag gagatcaagg acagagccac caccctgtac
      481 gagatcaccg gcaaggactg ggacagaaag gaggtgctgc tgaaggtgct gaagagaatc
      541 agcgagaacc tgaagaagtt caaggagaag agcttcaagg agttcaaggg caagatcgag
      601 agcaagatgc tgtacctggg cgaggaggtg aagctgctgg gcgagggcaa gatcaccggc
      661 aagctggtgg gcctgagcga gaagggcggc gccctgatcc tgaccgagga gggcatcaag
      721 gagatcctga gcggcgagtt cagcctgaga agaagcttaa ttaacatctt ttacccatac
      781 gatgttcctg actatgcggg ctatccctat gacgtcccgg actatgcagg atcctatcca
      841 tatgacgttc cagattacgc tgctcagtgc tgaggcgcgc cacttctaaa taagcgaatt
      901 tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat
      961 tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt
     1021 aggtcaggtt gctttctcag gtatagtatg aggtcgctct tattgaccac acctctaccg
     1081 gcagatccgc tagggataac agggtaatat agatctgttt agcttgcctc gtccccgccg
     1141 ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct tgacgtgcgc
     1201 agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc ccatgtataa
     1261 tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg
     1321 ctgcagacct gcgagcaggg aaacgctccc ctcacagacg cgttgaattg tccccacgcc
     1381 gcgcccctgt agagaaatat aaaaggttag gatttgccac tgaggttctt ctttcatata
     1441 cttcctttta aaatcttgct aggatacagt tctcacatca catccgaaca taaacaacca
     1501 tgggtaagga aaagactcac gtttcgaggc cgcgattaaa ttccaacatg gatgctgatt
     1561 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     1621 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     1681 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     1741 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     1801 gcaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     1861 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     1921 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     1981 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     2041 ataagctttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     2101 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     2161 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     2221 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     2281 ttcatttgat gctcgatgag tttttctaat cagtactgac aataaaaaga ttcttgtttt
     2341 caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt aatcaaatgt
     2401 tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga agttaagtgc
     2461 gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac tgctgtcgat
     2521 tcgatactaa cgccgccatc cagtttaaac gagctcgaat tcatcgatga tatcagatcc
     2581 actagtggcc tatgcggccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc
     2641 gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     2701 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2761 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2821 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2881 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2941 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3001 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3061 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3121 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3181 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3241 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3301 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     3361 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3421 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3481 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3541 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3601 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3661 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3721 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3781 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3841 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3901 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3961 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4021 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4081 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4141 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4201 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4261 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4321 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4381 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4441 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4501 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4561 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4621 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     4681 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     4741 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     4801 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     4861 tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg
     4921 cggctacaat taatacataa ccttatgtat catacacata cgatttaggt gacactatag
     4981 a
//
