LOCUS       sequence-438031-        7430 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FAP insert for C-terminally tagging genes of interest (optimized FAP
            for yeast expression + 2xMYC tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7430)
  AUTHORS   Oppenheimer KG, Hager NA, McAtee CK, Filiztekin E, Shang C, Warnick 
            JA, Bruchez MP, Brodsky JL, Prosser DC, Kwiatkowski AV, O'Donnell AF
  TITLE     Optimization of the fluorogen-activating protein tag for 
            quantitative protein trafficking and colocalization studies in S. 
            cerevisiae.
  JOURNAL   Mol Biol Cell. 2024 Jul 1;35(7):mr5. doi: 10.1091/mbc.E24-04-0174. 
            Epub 2024 May 29.
  PUBMED    38809589
REFERENCE   2  (bases 1 to 7430)
  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: "Mol Biol 
            Cell."; date: "2024-07-1"; volume: "35(7):mr5. doi"; pages: " 
            10.1091/mbc.E24-04-0174. Epub 2024 May 29"
COMMENT     Sequence Label: pRS415-MET25pr-CFAPoptim
FEATURES             Location/Qualifiers
     source          1..7430
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(167..186)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     286..308
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(346..364)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    406..909
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        946..1050
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1051..1119,1120..1911)
                     /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     complement(1269..1288)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2082..2670
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2571..2590
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2824..2841
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2958..2979
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2994..2999,3000..3017,3018..3024)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3032..3048
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3037..3059
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3056..3072
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3056..3072
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3091..3111
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3093..3111
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3162..3511
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     3512..3528
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3512..3528
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3562..3578)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3563..3579)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     CDS             3602..4321
                     /codon_start=1
                     /product="fluorogen-activating protein composed of a dimer 
                     of the L5-MG single-chain antibody fragment (Szent-Gyorgyi 
                     et al., 2008)"
                     /label=FAP-alpha-2
                     /note="binds malachite green derivatives to produce red 
                     fluorescence"
                     /translation="QAVVTQEPSVTVSPGGTVILTCGSGTGAVTSGHYANWFQQKPGQA
                     PRALIFDTDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSLSDVDGYLFGGGT
                     QLTVLSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSVTVSPGGTVILTCGSGTGAVTSGH
                     YANWFQQKPGQAPRALIFDTDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSL
                     SDVDGYLFGGGTQLTVLS"
     CDS             4322..4351
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4352..4381
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     terminator      4394..4641
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4410..4428)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4659..4678)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4660..4678)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4688..4705)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4688..4704)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4697..4719)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4845..5300
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4932..4951)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5142..5163
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5601..6008
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             6009..7103
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
ORIGIN
        1 aaaaaaaaaa aatacaacaa ataaaaaaca ctcaatgacc tgaccatttg atggagttta
       61 agtcaatacc ttcttgaacc atttcccata atggtgaaag ttccctcaag aattttactc
      121 tgtcagaaac ggccttaacg acgtagtcga tatggtgcac tctcagtaca atctgctctg
      181 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
      241 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
      301 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
      361 tatttttata ggttaatgtc atgataataa tggtttctta ggacggatcg cttgcctgta
      421 acttacacgc gcctcgtatc ttttaatgat ggaataattt gggaatttac tctgtgttta
      481 tttattttta tgttttgtat ttggatttta gaaagtaaat aaagaaggta gaagagttac
      541 ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt caacaaaaag cgtactttac
      601 atatatattt attagacaag aaaagcagat taaatagata tacattcgat taacgataag
      661 taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta cacagacaag atgaaacaat
      721 tcggcattaa tacctgagag caggaagagc aagataaaag gtagtatttg ttggcgatcc
      781 ccctagagtc ttttacatct tcggaaaaca aaaactattt tttctttaat ttcttttttt
      841 actttctatt tttaatttat atatttatat taaaaaattt aaattataat tatttttata
      901 gcacgtgatg aaaaggaccc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
      961 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1021 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1081 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1141 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1201 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1261 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     1321 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     1381 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     1441 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     1501 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     1561 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     1621 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     1681 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     1741 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     1801 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     1861 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     1921 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     1981 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2041 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2101 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2161 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2221 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2281 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2341 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2401 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2461 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2521 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2581 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2641 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2701 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2761 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     2821 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     2881 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2941 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     3001 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     3061 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     3121 ctggagctcc ggatgcaagg gttcgaatcc cttagctctc attatttttt gctttttctc
     3181 ttgaggtcac atgatcgcaa aatggcaaat ggcacgtgaa gctgtcgata ttggggaact
     3241 gtggtggttg gcaaatgact aattaagtta gtcaaggcgc catcctcatg aaaactgtgt
     3301 aacataataa ccgaagtgtc gaaaaggtgg caccttgtcc aattgaacac gctcgatgaa
     3361 aaaaataaga tatatataag gttaagtaaa gcgtctgtta gaaaggaagt ttttcctttt
     3421 tcttgctctc ttgtcttttc atctactatt tccttcgtgt aatacagggt cgtcagatac
     3481 atagatacaa ttctattacc cccatccata ctctagaact agtggatccc ccgggctgca
     3541 ggaattcgat atcaagctta tcgataccgt cgacctcgac gcgggtgcgg gtgcgggtat
     3601 gcaggccgtc gttacccaag aacctagtgt taccgttagc ccaggtggta ctgttatact
     3661 tacttgtgga agtggtacgg gtgccgtcac atctggtcat tatgcaaatt ggtttcaaca
     3721 aaaaccagga caagctccaa gagctttgat ttttgatact gataagaagt attcttggac
     3781 cccaggtaga ttttctggat ctttgctggg agcaaaggca gctttgacaa tatcagatgc
     3841 tcagcctgag gacgaagccg agtattactg ttctcttagc gacgtggatg gctacttgtt
     3901 tggcggtgga acacaactga cggttctgtc cggtggtggc ggctctggtg gcggtggcag
     3961 cggcggtggt ggttccggag gcggcggttc tcaggctgtg gtgactcagg agccgtcagt
     4021 gactgtgtcc ccaggaggga cagtcattct cacttgtggc tccggcactg gagctgtcac
     4081 cagtggtcat tatgccaact ggttccagca gaagcctggc caagccccca gggcacttat
     4141 atttgacacc gacaagaagt attcctggac ccctggccga ttctcaggct ccctccttgg
     4201 ggccaaggct gccctgacca tctcggatgc gcagcctgaa gatgaggctg agtattactg
     4261 ttcgctctcc gacgttgacg gttatctgtt cggaggaggc acccagctga ccgtcctctc
     4321 cgagcagaag ctgatcagcg aggaggacct ggagcagaag ctgatcagcg aggaggacct
     4381 gtaacgcctc gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca
     4441 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt
     4501 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
     4561 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
     4621 cgctcgaagg ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg
     4681 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     4741 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     4801 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg
     4861 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4921 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4981 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5041 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5101 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5161 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     5221 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5281 aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5341 tcacaccgca tatcgaccgg tcgaggagaa cttctagtat atctacatac ctaatattat
     5401 tgccttatta aaaatggaat cccaacaatt acatcaaaat ccacattctc ttcaaaatca
     5461 attgtcctgt acttccttgt tcatgtgtgt tcaaaaacgt tatatttata ggataattat
     5521 actctatttc tcaacaagta attggttgtt tggccgagcg gtctaaggcg cctgattcaa
     5581 gaaatatctt gaccgcagtt aactgtggga atactcaggt atcgtaagat gcaagagttc
     5641 gaatctctta gcaaccatta tttttttcct caacataacg agaacacaca ggggcgctat
     5701 cgcacagaat caaattcgat gactggaaat tttttgttaa tttcagaggt cgcctgacgc
     5761 atataccttt ttcaactgaa aaattgggag aaaaaggaaa ggtgagagcg ccggaaccgg
     5821 cttttcatat agaatagaga agcgttcatg actaaatgct tgcatcacaa tacttgaagt
     5881 tgacaatatt atttaaggac ctattgtttt ttccaatagg tggttagcaa tcgtcttact
     5941 ttctaacttt tcttaccttt tacatttcag caatatatat atatatattt caaggatata
     6001 ccattctaat gtctgcccct aagaagatcg tcgttttgcc aggtgaccac gttggtcaag
     6061 aaatcacagc cgaagccatt aaggttctta aagctatttc tgatgttcgt tccaatgtca
     6121 agttcgattt cgaaaatcat ttaattggtg gtgctgctat cgatgctaca ggtgttccac
     6181 ttccagatga ggcgctggaa gcctccaaga aggctgatgc cgttttgtta ggtgctgtgg
     6241 gtggtcctaa atggggtacc ggtagtgtta gacctgaaca aggtttacta aaaatccgta
     6301 aagaacttca attgtacgcc aacttaagac catgtaactt tgcatccgac tctcttttag
     6361 acttatctcc aatcaagcca caatttgcta aaggtactga cttcgttgtt gtcagagaat
     6421 tagtgggagg tatttacttt ggtaagagaa aggaagacga tggtgatggt gtcgcttggg
     6481 atagtgaaca atacaccgtt ccagaagtgc aaagaatcac aagaatggcc gctttcatgg
     6541 ccctacaaca tgagccacca ttgcctattt ggtccttgga taaagctaat gttttggcct
     6601 cttcaagatt atggagaaaa actgtggagg aaaccatcaa gaacgaattc cctacattga
     6661 aggttcaaca tcaattgatt gattctgccg ccatgatcct agttaagaac ccaacccacc
     6721 taaatggtat tataatcacc agcaacatgt ttggtgatat catctccgat gaagcctccg
     6781 ttatcccagg ttccttgggt ttgttgccat ctgcgtcctt ggcctctttg ccagacaaga
     6841 acaccgcatt tggtttgtac gaaccatgcc acggttctgc tccagatttg ccaaagaata
     6901 aggtcaaccc tatcgccact atcttgtctg ctgcaatgat gttgaaattg tcattgaact
     6961 tgcctgaaga aggtaaggcc attgaagatg cagttaaaaa ggttttggat gcaggtatca
     7021 gaactggtga tttaggtggt tccaacagta ccaccgaagt cggtgatgct gtcgccgaag
     7081 aagttaagaa aatccttgct taaaaagatt ctcttttttt atgatatttg tacataaact
     7141 ttataaatga aattcataat agaaacgaca cgaaattaca aaatggaata tgttcatagg
     7201 gtagacgaaa ctatatacgc aatctacata catttatcaa gaaggagaaa aaggaggatg
     7261 taaaggaata caggtaagca aattgatact aatggctcaa cgtgataagg aaaaagaatt
     7321 gcactttaac attaatattg acaaggagga gggcaccaca caaaaagtta ggtgtaacag
     7381 aaaatcatga aactatgatt cctaatttat atattggagg attttctcta
//
