LOCUS       sequence-361815-        5120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR-based tagging of yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5120)
  AUTHORS   Lamb AK, Di Pietro SM
  TITLE     Utilizing chemically induced dimerization of FKBP to analyze 
            endocytosis by live-cell imaging in budding yeast.
  JOURNAL   STAR Protoc. 2022 Apr 14;3(2):101323. doi: 
            10.1016/j.xpro.2022.101323. eCollection 2022 Jun 17.
  PUBMED    35496798
REFERENCE   2  (bases 1 to 5120)
  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: "STAR 
            Protoc."; date: "2022-04-14"; volume: "3(2):101323. doi"; pages: " 
            10.1016/j.xpro.2022.101323. eCollection 2022 Jun 17"
COMMENT     Sequence Label: pFA6a-FKBP-GFP-HIS3MX6
FEATURES             Location/Qualifiers
     source          1..5120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             78..398
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             414..1124
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(441..469)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1064..1084
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1148..1335
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1203..1227)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        1410..1753
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1754..2452
                     /codon_start=1
                     /product="S. kluyveri imidazoleglycerol phosphate 
                     dehydratase"
                     /label=k-HIS3
                     /translation="MAEPAQKKQKQTVQERKAFISRITNETKIQIAISLNGGYIQIKDS
                     ILPAKKDDDVASQATQSQVIDIHTGVGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHT
                     TEDCGIALGQAFKEAMGAVRGVKRFGTGFAPLDEALSRAVVDLSSRPFAVIDLGLKREM
                     IGDLSTEMIPHFLESFAEAARITLHVDCLRGFNDHHRSESAFKALAVAIREAISSNGTN
                     DVPSTKGVLM"
     terminator      2456..2653
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2757..2776)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2758..2776)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2863..2880)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3034..3622)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3114..3133)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3793..4584,4585..4653))
                     /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     4416..4435
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4654..4758)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4826..4844
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4882..4904)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5004..5023
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5104..5120,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5104..5120,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggttaatta
       61 acggaggagg aggaagcgga gtgcaggtgg agactatctc cccaggagac gggcgcacct
      121 tccccaagcg cggccagacc tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga
      181 aagttgattc ctcccgggac agaaacaagc cctttaagtt tatgctaggc aagcaggagg
      241 tgatccgagg ctgggaagaa ggggttgccc agatgagtgt gggtcagaga gccaaactga
      301 ctatatctcc agattatgcc tatggtgcca ctgggcaccc aggcatcatc ccaccacatg
      361 ccactctcgt cttcgatgtg gagcttctaa aactggaagg aggaggagga agcagtaaag
      421 gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg
      481 ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc
      541 ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt
      601 tcacttatgg tgttcaatgc ttttcaagat acccagatca tatgaaacgg catgactttt
      661 tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc aaagatgacg
      721 ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg
      781 agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa ttggaataca
      841 actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga atcaaagtta
      901 acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac cattatcaac
      961 aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtccacac
     1021 aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt cttgagtttg
     1081 taacagctgc tgggattaca catggcatgg atgaactata caaatagggc gcgccacttc
     1141 taaataagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat
     1201 aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag
     1261 taactctttc ctgtaggtca ggttgctttc tcaggtatag tatgaggtcg ctcttattga
     1321 ccacacctct accggcagat ccgctaggga taacagggta atatagatct gtttagcttg
     1381 cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc ctccttgaca
     1441 gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt tagcccatac
     1501 atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg cgaagcaaaa
     1561 attacggctc ctcgctgcag acctgcgagc agggaaacgc tcccctcaca gacgcgttga
     1621 attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg ccactgaggt
     1681 tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac atcacatccg
     1741 aacataaaca accatggcag aaccagccca aaaaaagcaa aaacaaactg ttcaggagcg
     1801 caaggcgttt atctcccgta tcactaatga aactaaaatt caaatcgcta tttcgctgaa
     1861 tggtggttat attcaaataa aagattcgat tcttcctgca aagaaggatg acgatgtagc
     1921 ttcccaagct actcagtcac aggtcatcga tattcacaca ggtgttggct ttttggatca
     1981 tatgatccat gcgttggcaa aacactctgg ttggtctctt attgttgaat gtattggtga
     2041 cctgcacatt gacgatcacc atactaccga agattgcggt atcgcattag ggcaagcgtt
     2101 caaagaagca atgggtgctg tccgtggtgt aaaaagattc ggtactgggt tcgcaccatt
     2161 ggatgaggcg ctatcacgtg ccgtagtcga tttatctagt agaccatttg ctgtaatcga
     2221 ccttggattg aagagagaga tgattggtga tttatccact gaaatgattc cacacttttt
     2281 ggaaagtttc gcggaggcgg ccagaattac tttgcatgtt gattgtctga gaggtttcaa
     2341 cgatcaccac agaagtgaga gtgcgttcaa ggctttggct gttgccataa gagaagctat
     2401 ttctagcaat ggcaccaatg acgttccctc aaccaaaggt gttttgatgt gaagtactga
     2461 caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt
     2521 tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg
     2581 cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct
     2641 ggtcgctata ctgctgtcga ttcgatacta acgccgccat ccagtttaaa cgagctcgaa
     2701 ttcatcgatg atatcagatc cactagtggc ctatgcggcc gcggatctgc cggtctccct
     2761 atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg
     2821 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2881 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2941 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3001 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3061 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3121 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3181 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3241 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3301 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3361 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3421 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3481 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3541 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3601 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3661 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3721 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3781 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3841 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3901 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3961 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4021 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4081 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4141 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4201 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4261 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4321 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4381 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4441 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4501 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4561 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4621 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4681 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4741 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     4801 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     4861 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     4921 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     4981 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     5041 ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
     5101 acgatttagg tgacactata
//
