LOCUS       sequence-451852-        5245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To insert HA-tagged biotin ligase, BirA, at the C-terminus of genes;
            yeast BioID with KanMX6 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5245)
  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 5245)
  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-BirA-HA3-KanMX6
FEATURES             Location/Qualifiers
     source          1..5245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(61..408,409..411,412..1020)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1036..1125
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      1158..1345
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1213..1237)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1420..2776
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1420..1763
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1764..2573
                     /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(1831..1850)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2579..2776
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2880..2899)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2881..2899)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2986..3003)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3157..3745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3237..3256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3916..4707,4708..4776))
                     /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     4539..4558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4777..4881)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4949..4967
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5005..5027)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5127..5146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5227..5245
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5227..5244
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 acgcggccgc cagctgaagc ttcgtacgct gcaggtcgac ggatccccgg gttaattaac
       61 aaggacaaca ccgtgcccct gaagctgatc gccctgctgg ccaacggcga gttccactct
      121 ggcgagcagc tgggagagac cctgggaatg agcagagccg ccatcaacaa gcacatccag
      181 acactgagag actggggagt ggacgtgttc accgtgcctg gcaagggcta cagcctgcct
      241 gagcctatcc agctgctgaa cgccaagcag atcctgggac agctggatgg cggaagcgtg
      301 gccgtgctgc ctgtgatcga ctccaccaat cagtacctgc tggacagaat cggagagctg
      361 aagtccggcg acgcctgcat cgccgagtac cagcaggctg gcagaggagg cagaggacgg
      421 aagtggttca gcccattcgg agccaacctg tacctgtcca tgttctggag actggagcag
      481 ggacctgctg ctgccatcgg actgagtctg gtgatcggaa tcgtgatggc cgaggtgctg
      541 agaaagctgg gagccgacaa ggtgagagtg aagtggccta atgacctgta cctccaggac
      601 cgcaagctgg ctggcatcct ggtggagctg acaggcaaga caggcgatgc cgctcagatc
      661 gtgatcggag ccggaatcaa catggccatg agaagagtgg aggagagcgt ggtgaaccag
      721 ggctggatca ccctgcagga ggctggcatc aacctggacc ggaacaccct ggccgccatg
      781 ctgatcagag agctgagagc cgctctggag ctgttcgagc aggagggact ggctccttac
      841 ctgagcagat gggagaagct ggacaacttc atcaacagac ctgtgaagct gatcatcggc
      901 gacaaggaaa tcttcggcat ctccagagga atcgacaagc agggagctct gctgctggag
      961 caggacggaa tcatcaagcc ctggatgggc ggagaaatct ccctgagaag cgcagagaag
     1021 ttaattaaca tcttttaccc atacgatgtt cctgactatg cgggctatcc ctatgacgtc
     1081 ccggactatg caggatccta tccatatgac gttccagatt acgctgctca gtgctgaggc
     1141 gcgccacttc taaataagcg aatttcttat gatttatgat ttttattatt aaataagtta
     1201 taaaaaaaat aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct
     1261 tattcttgag taactctttc ctgtaggtca ggttgctttc tcaggtatag tatgaggtcg
     1321 ctcttattga ccacacctct accggcagat ccgctaggga taacagggta atatagatct
     1381 gtttagcttg cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc
     1441 ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt
     1501 tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg
     1561 cgaagcaaaa attacggctc ctcgctgcag acctgcgagc agggaaacgc tcccctcaca
     1621 gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg
     1681 ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac
     1741 atcacatccg aacataaaca accatgggta aggaaaagac tcacgtttcg aggccgcgat
     1801 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     1861 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     1921 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     1981 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     2041 ggttactcac cactgcgatc cccggcaaaa cagcattcca ggtattagaa gaatatcctg
     2101 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     2161 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     2221 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     2281 ttgaacaagt ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca
     2341 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     2401 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     2461 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     2521 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagtac
     2581 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt
     2641 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat
     2701 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat
     2761 gctggtcgct atactgctgt cgattcgata ctaacgccgc catccagttt aaacgagctc
     2821 gaattcatcg atgatatcag atccactagt ggcctatgcg gccgcggatc tgccggtctc
     2881 cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca
     2941 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3001 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3061 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3121 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3181 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3241 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3301 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3361 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3421 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3481 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3541 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3601 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3661 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3721 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3781 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3841 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3901 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3961 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4021 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4081 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4141 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4201 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4261 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4321 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4381 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4441 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4501 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4561 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4621 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4681 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4741 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4801 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4861 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     4921 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     4981 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     5041 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     5101 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     5161 tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca
     5221 catacgattt aggtgacact ataga
//
