LOCUS       sequence-451854-        5132 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To insert HA-tagged biotin ligase, BirA, at the C-terminus of genes;
            yeast BioID, HIS3MX6 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5132)
  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 5132)
  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-HIS3MX6
FEATURES             Location/Qualifiers
     source          1..5132
                     /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"
     promoter        1420..1763
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1764..2462
                     /codon_start=1
                     /product="S. kluyveri imidazoleglycerol phosphate 
                     dehydratase"
                     /label=k-HIS3
                     /translation="MAEPAQKKQKQTVQERKAFISRITNETKIQIAISLNGGYIQIKDS
                     ILPAKKDDDVASQATQSQVIDIHTGVGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHT
                     TEDCGIALGQAFKEAMGAVRGVKRFGTGFAPLDEALSRAVVDLSSRPFAVIDLGLKREM
                     IGDLSTEMIPHFLESFAEAARITLHVDCLRGFNDHHRSESAFKALAVAIREAISSNGTN
                     DVPSTKGVLM"
     terminator      2466..2663
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2767..2786)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2768..2786)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2873..2890)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3044..3632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3124..3143)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3803..4594,4595..4663))
                     /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     4426..4445
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4664..4768)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4836..4854
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4892..4914)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5014..5033
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5114..5132
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5114..5131
                     /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 accatggcag aaccagccca aaaaaagcaa aaacaaactg
     1801 ttcaggagcg caaggcgttt atctcccgta tcactaatga aactaaaatt caaatcgcta
     1861 tttcgctgaa tggtggttat attcaaataa aagattcgat tcttcctgca aagaaggatg
     1921 acgatgtagc ttcccaagct actcagtcac aggtcatcga tattcacaca ggtgttggct
     1981 ttttggatca tatgatccat gcgttggcaa aacactctgg ttggtctctt attgttgaat
     2041 gtattggtga cctgcacatt gacgatcacc atactaccga agattgcggt atcgcattag
     2101 ggcaagcgtt caaagaagca atgggtgctg tccgtggtgt aaaaagattc ggtactgggt
     2161 tcgcaccatt ggatgaggcg ctatcacgtg ccgtagtcga tttatctagt agaccatttg
     2221 ctgtaatcga ccttggattg aagagagaga tgattggtga tttatccact gaaatgattc
     2281 cacacttttt ggaaagtttc gcggaggcgg ccagaattac tttgcatgtt gattgtctga
     2341 gaggtttcaa cgatcaccac agaagtgaga gtgcgttcaa ggctttggct gttgccataa
     2401 gagaagctat ttctagcaat ggcaccaatg acgttccctc aaccaaaggt gttttgatgt
     2461 gaagtactga caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt
     2521 atattgtagt tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg
     2581 acatcatctg cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta
     2641 tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta acgccgccat ccagtttaaa
     2701 cgagctcgaa ttcatcgatg atatcagatc cactagtggc ctatgcggcc gcggatctgc
     2761 cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa
     2821 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2881 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2941 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3001 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3061 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3121 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3181 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3241 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3301 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3361 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3421 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3481 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3541 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3601 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3661 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3721 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3781 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3841 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3901 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3961 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4021 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4081 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4141 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4201 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4261 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4321 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4381 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4441 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4501 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4561 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4621 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4681 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4741 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     4801 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     4861 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     4921 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     4981 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     5041 tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta
     5101 tcatacacat acgatttagg tgacactata ga
//
