LOCUS       sequence-454154-        4868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To insert HA-tagged biotin ligase, BioID2, 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 4868)
  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 4868)
  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-HIS3MX6
FEATURES             Location/Qualifiers
     source          1..4868
                     /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"
     promoter        1156..1499
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1500..2198
                     /codon_start=1
                     /product="S. kluyveri imidazoleglycerol phosphate 
                     dehydratase"
                     /label=k-HIS3
                     /translation="MAEPAQKKQKQTVQERKAFISRITNETKIQIAISLNGGYIQIKDS
                     ILPAKKDDDVASQATQSQVIDIHTGVGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHT
                     TEDCGIALGQAFKEAMGAVRGVKRFGTGFAPLDEALSRAVVDLSSRPFAVIDLGLKREM
                     IGDLSTEMIPHFLESFAEAARITLHVDCLRGFNDHHRSESAFKALAVAIREAISSNGTN
                     DVPSTKGVLM"
     terminator      2202..2399
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2503..2522)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2504..2522)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2609..2626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2780..3368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2860..2879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3539..4330,4331..4399))
                     /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     4162..4181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4400..4504)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4572..4590
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4628..4650)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4750..4769
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4850..4868
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4850..4867
                     /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 tggcagaacc agcccaaaaa aagcaaaaac aaactgttca ggagcgcaag gcgtttatct
     1561 cccgtatcac taatgaaact aaaattcaaa tcgctatttc gctgaatggt ggttatattc
     1621 aaataaaaga ttcgattctt cctgcaaaga aggatgacga tgtagcttcc caagctactc
     1681 agtcacaggt catcgatatt cacacaggtg ttggcttttt ggatcatatg atccatgcgt
     1741 tggcaaaaca ctctggttgg tctcttattg ttgaatgtat tggtgacctg cacattgacg
     1801 atcaccatac taccgaagat tgcggtatcg cattagggca agcgttcaaa gaagcaatgg
     1861 gtgctgtccg tggtgtaaaa agattcggta ctgggttcgc accattggat gaggcgctat
     1921 cacgtgccgt agtcgattta tctagtagac catttgctgt aatcgacctt ggattgaaga
     1981 gagagatgat tggtgattta tccactgaaa tgattccaca ctttttggaa agtttcgcgg
     2041 aggcggccag aattactttg catgttgatt gtctgagagg tttcaacgat caccacagaa
     2101 gtgagagtgc gttcaaggct ttggctgttg ccataagaga agctatttct agcaatggca
     2161 ccaatgacgt tccctcaacc aaaggtgttt tgatgtgaag tactgacaat aaaaagattc
     2221 ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat
     2281 caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt
     2341 taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgc
     2401 tgtcgattcg atactaacgc cgccatccag tttaaacgag ctcgaattca tcgatgatat
     2461 cagatccact agtggcctat gcggccgcgg atctgccggt ctccctatag tgagtcgtat
     2521 taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2581 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2941 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3181 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3301 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3361 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3541 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3601 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3661 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3721 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3781 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3841 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3901 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3961 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4021 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4081 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4141 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4201 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4261 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4321 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4381 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4441 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4501 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     4561 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     4621 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     4681 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     4741 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt
     4801 tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac
     4861 actataga
//
