LOCUS       sequence-429383-        7645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative fluorescent reporter for amino acid uptake sensing 
            pathway, comprising mKate2 driven by the GNP1 promoter within the 
            Easy clone vector, requires NotI digestion for integration in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7645)
  AUTHORS   Paukstyte J, Tena EC, Saarikangas J
  TITLE     A dual reporter system for intracellular and extracellular amino 
            acid sensing in budding yeast.
  JOURNAL   Mol Biol Cell. 2025 Apr 2:mbcE24040162. doi: 
            10.1091/mbc.E24-04-0162.
  PUBMED    40172974
REFERENCE   2  (bases 1 to 7645)
  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. 2025 Apr 2:mbcE24040162. doi: 10.1091/mbc.E24-04-0162."
COMMENT     Sequence Label: pCfB2197-PrGNP1-mK2-NatMX
FEATURES             Location/Qualifiers
     source          1..7645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      12..201
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    249..282
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            337..1456
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        337..680
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             681..1253
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1259..1456
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1519..1552
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1595..1614)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1596..1614)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1624..1640)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1633..1655)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2332..2365)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(2835..2851)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2950..2967)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3121..3709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3201..3220)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3831..4622,4623..4691))
                     /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     4454..4473
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4692..4783)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4825..4844
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4850..4869
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5076..5109
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      complement(5702..5867)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5788..5812
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 cgcgtgcatt catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc
       61 cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt
      121 cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag
      181 aaggttttgg gacgctcgaa gatcgcgtca gctgaagctt cgtacgctgc aggtcgacaa
      241 cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtcta gagatctgtt
      301 tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca gaataccctc
      361 cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag
      421 cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga
      481 agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc cctcacagac
      541 gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca
      601 ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc
      661 acatccgaac ataaacaacc atgggtacca ctcttgacga cacggcttac cggtaccgca
      721 ccagtgtccc gggggacgcc gaggccatcg aggcactgga tgggtccttc accaccgaca
      781 ccgttttccg cgtcaccgcc accggggacg gcttcaccct gcgggaggtg ccggtggacc
      841 cgcccctgac caaggtgttc cccgacgacg aatcggacga cgaatcggac gacggggagg
      901 acggcgaccc ggactcccgg acgttcgtcg cgtacgggga cgacggcgac ctggcgggct
      961 tcgtggtcgt ctcgtactcc ggctggaacc gccggctgac cgtcgaggac atcgaggtcg
     1021 ccccggagca ccgggggcac ggggtcgggc gcgcgttgat ggggctcgcg acggagttcg
     1081 cccgcgagcg gggcgccggg cacctctggc tggaggtcac caacgtcaac gcaccggcga
     1141 tccacgcgta ccggcggatg gggttcaccc tctgcggcct ggacaccgcc ctgtacgacg
     1201 gcaccgcctc ggacggcgag caggcgctct acatgagcat gccctgcccc taatcagtac
     1261 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt
     1321 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat
     1381 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat
     1441 gctggtcgct atactgctgt cgattcgata ctaacgccgc catccagtgt cgaaaacgag
     1501 ctctcgagaa cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtgat
     1561 atcagatcca ctagtggcct atgcacccaa ttcgccctat agtgagtcgt attacgcgcg
     1621 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccctgcagg
     1681 actagtgctg aggcattaat cttcccgtga atcaactgca cataccttaa taactagttc
     1741 agtttagtgc tctgtctgag tgactgtaat aataaatttt acaagccact tctcatgaca
     1801 tatattggta agtaacttca tcaatactaa ttagtctttg ccggttaccc atctggcccc
     1861 tgacttgcga tgcttaggaa gttccatact cgcggctctt cccaacagta gcacatccgt
     1921 gaaacttctg gcgctattca ttatgcagta ccaggacaag aagttaaaaa aaaagctctg
     1981 ttacaagttc aatggtggtg caaggattga agttattatc caggaggcac ggatgcaaaa
     2041 gatagaggac aaattaattt ccttaaaatt caaaatgctc attattgtca cgctgtatga
     2101 gcatttggtg aagatttcac tgggaaatgt tgcaataatt tgataatcgt tcgtattgga
     2161 tgaaactgta acatcatctg tttattaagt atccgtgtta ttagtatatc atcacatacg
     2221 gtgtaagaag ataacataaa gattgagaaa cagtcatcaa atataatgga agctgaaatg
     2281 cgaggattga ttaagacctc agcgcggccg caaatttaaa taaaatgaag tgaagttcct
     2341 atactttcta gagaatagga acttctatag tgagtcgaat aagggcgaca caaaatttat
     2401 tctaaatgca taataaatac tgataacatc ttatagtttg tattatattt tgtattatcg
     2461 ttgacatgta taattttgat atcaaaaact gattttccct ttattatttt cgagatttat
     2521 tttcttaatt ctctttaaca aactagaaat attgtatata caaaaaatca taaataatag
     2581 atgaatagtt taattatagg tgttcatcaa tcgaaaaagc aacgtatctt atttaaagtg
     2641 cgttgctttt ttctcattta taaggttaaa taattctcat atatcaagca aagtgacagg
     2701 cgcccttaaa tattctgaca aatgctcttt ccctaaactc cccccataaa aaaacccgcc
     2761 gaagcgggtt tttacgttat ttgcggatta acgattactc gttatcagaa ccgcccaggg
     2821 ggcccgagct taagactggc cgtcgtttta caacacagaa agagtttgta gaaacgcaaa
     2881 aaggccatcc gtcaggggcc ttctgcttag tttgatgcct ggcagttccc tactctcgcc
     2941 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3001 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3061 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3121 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3181 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3241 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3301 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3361 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3421 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3481 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtgggc
     3541 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3601 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3661 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3721 gatcttttct acggggtctg acgctcagtg gaacgacgcg cgcgtaactc acgttaaggg
     3781 attttggtca tgagcttgcg ccgtcccgtc aagtcagcgt aatgctctgc ttaccaatgc
     3841 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3901 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagcgctgcg
     3961 atgataccgc gagaaccacg ctcaccggct ccggatttat cagcaataaa ccagccagcc
     4021 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4081 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4141 atcgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4201 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4261 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4321 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4381 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4441 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4501 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4561 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4621 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4681 tgaatactca tattcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4741 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt cagtgttaca
     4801 accaattaac caattctgaa cattatcgcg agcccattta tacctgaata tggctcataa
     4861 caccccttgt ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     4921 agaagtgaaa cgccgtagcg ccgatggtag tgtggggact ccccatgcga gagtagggaa
     4981 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgcccgggct
     5041 aattatgggg tgtcgccctt attcgactct atagtgaagt tcctattctc tagaaagtat
     5101 aggaacttct gaagtgggga tttaaatgcg gccgcgctga gggtttaatg ttgagctctg
     5161 tccttcatgg actttttgga ccacttcttc ttggacttct taccaccagc aagagcggca
     5221 gcggctttgg cagctttaga caattgttgc tttggaggca tgttatatta tgttctgaga
     5281 cgtaagaaag ggtgaaaatt gatgttagtg tcaaaaatta tattacaaaa tacgcagaga
     5341 tattctagtt cctttgatga atgaatcttt cagaaaaaaa agtcaaagca aaagcaaaat
     5401 ggcctgcaga ctaaactgta tggtggtctt ggaatgataa agatctgttt aatagattta
     5461 gtagatacaa tagcacatct cattacccag ttatgattga cgtcattctg agttacaatg
     5521 atcttagcga cctgtgctca ttatttgctc cactaattct aattttcctc gcctttcata
     5581 tttcgtatct ttattctata tcctaaaatt tttttggcaa atcccagatt tggctttgat
     5641 tttggcatcg gttcggttct ttcattaagt cctcagcgag ctcgcatgga atgcgtgcga
     5701 tgagcgacct catgctatac ctgagaaagc aacctgacct acaggaaaga gttactcaag
     5761 aataagaatt ttcgttttaa aacctaagag tcactttaaa atttgtatac acttattttt
     5821 tttataactt atttaataat aaaaatcata aatcataaga aattcgctta tttagaagtg
     5881 tcaacaacgt atctaccaac ggaatgcgtg cgatcttgac ctctggctta gcttcttgat
     5941 tgacttctga gtccgacatc gctcgtgtat ttatttgtaa aactagaact gaactgggtc
     6001 cttctgtcct atatgctaca ggtggtaaca agaaacggga gcttggaaaa ggtaagaaaa
     6061 atcaactaaa agagtagaat gtgttcgttt gtttgattgt tggtgttttg ctcttttcct
     6121 gcttcctgaa caccttccga cggcgcgtgt tggcaaagct gggtaaccca acatattcat
     6181 atcacatgat caaagagttc ggcggcaaaa acgtgacatg cacgtgatgg aaacagcatg
     6241 gcttttttca gttttatttt tttttttcac tttcaaaagc attattcctt ggggactacg
     6301 taatcaacgc gttggtcatt tctttttcca acgcgcatga ctaacctact atgacgacgt
     6361 agccttaatt cggcattacc gtattatgcg acgtgcttgc gatccttgaa aacggcatca
     6421 ggatgcacaa aaacgtttca aaagtgcccc gccgtacggt atgcggcgcc aaaaaccggt
     6481 gaacatgaaa aagaaacggc acagggacca tcaaaaatag gaaaaaagaa taacgccgca
     6541 tgaaaagaaa gcggatggaa aggcacggca actcttagag atgaaggcga tgatgcgatg
     6601 gcagacggac aatgaaatgt aaatttgaag tttttttcgg aaatcggttt tagtgttcgt
     6661 atgtcagttt ggctcgggtt gtgcagtgtc gtttcttttt tgagtccctt cttcccccac
     6721 ctctttgatg ttagaaagaa tcggattctg tatatataac cgattttttt cacatttctg
     6781 ttgatttaaa catattcctt tcgtgcttct tcaaagttga gagttatagg tgctttatgt
     6841 tttttttttt ttgtgtaaga aaaaaaaact atcgctttct caagtagctt atataatatc
     6901 aaatattgca cattatctgt cataaaacaa tgggttctgg tggttctgaa ttgatcaaag
     6961 aaaacatgca catgaagttg tacatggaag gtactgttaa caaccatcat ttcaagtgta
     7021 cctctgaagg tgaaggtaaa ccatatgaag gtactcaaac catgagaatc aaagctgttg
     7081 aaggtggtcc attgccattt gcttttgata ttttggctac ctccttcatg tacggttcta
     7141 agactttcat taaccacacc caaggtatcc cagatttctt caaacaatct ttcccagaag
     7201 gtttcacttg ggaaagagtt actacttacg aagatggtgg tgttttgact gctactcaag
     7261 atacttcttt acaagacggt tgcttgatct acaacgttaa gattagaggt gtcaacttcc
     7321 catctaatgg tccagttatg caaaaaaaga ctttgggttg ggaagcttct actgaaacat
     7381 tatatccagc tgatggtggt ttggaaggta gagctgatat ggctttgaaa ttggttggtg
     7441 gtggtcattt gatctgcaac ttgaaaacta cctacagatc taagaagcca gccaagaatt
     7501 tgaaaatgcc aggtgtttac tacgtcgata gaagattgga aagaatcaaa gaagccgaca
     7561 aagaaaccta cgttgaacaa cacgaagttg ctgttgctag atattgtgat ttgccatcca
     7621 aattaggtca tagaggatct tgaat
//
