LOCUS       sequence-323177-        8283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast expression of the mutant #4_1m for the RNA-based sensor of 
            fructose-1,6-bisphosphate #4_1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8283)
  AUTHORS   Ortega AD, Takhaveev V, Vedelaar SR, Long Y, Mestre-Farras N, 
            Incarnato D, Ersoy F, Olsen LF, Mayer G, Heinemann M
  TITLE     A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that 
            reports glycolytic flux.
  JOURNAL   Cell Chem Biol. 2021 Apr 22. pii: S2451-9456(21)00161-6. doi: 
            10.1016/j.chembiol.2021.04.006.
  PUBMED    33915105
REFERENCE   2  (bases 1 to 8283)
  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: "Cell Chem 
            Biol. 2021 Apr 22. pii: S2451-9456(21)00161-6. doi: 
            10.1016/j.chembiol.2021.04.006."
COMMENT     Sequence Label: pFBP-4_1m.mutant
FEATURES             Location/Qualifiers
     source          1..8283
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1963..1982
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1963..1981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1995..2392
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     terminator      3148..3395
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3164..3182)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     3860..3879
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        3960..4356
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             4416..5132
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGIIHGMDELYK"
     terminator      5275..5462
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5330..5354)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(5510..5528)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5549..5565)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5549..5565)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5562..5584)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5573..5589
                     /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)."
     promoter        complement(join(5597..5603,5604..5621,5622..5627))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5642..5663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5780..5797)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5951..6539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6031..6050)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6710..7501,7502..7570))
                     /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     7333..7352
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7571..7675)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7712..8215
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     8256..8274
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcggggg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     1981 ggcgaattgg agctcatagc ttcaaaatgt ttctactcct tttttactct tccagatttt
     2041 ctcggactcc gcgcatcgcc gtaccacttc aaaacaccca agcacagcat actaaatttc
     2101 ccctctttct tcctctaggg tgtcgttaat tacccgtact aaaggtttgg aaaagaaaaa
     2161 agagaccgcc tcgtttcttt ttcttcgtcg aaaaaggcaa taaaaatttt tatcacgttt
     2221 ctttttcttg aaaatttttt tttgattttt ttctctttcg atgacctccc attgatattt
     2281 aagttaataa acggtcttca atttctcaag tttcagtttc atttttcttg ttctattaca
     2341 acttttttta cttcttgctc attagaaaga aagcatagca atctaatcta agttttgccg
     2401 cgggaaataa tgtctatggt tagtaaagga gaagaaaata acatggcaat cattaaggag
     2461 ttcatgagat tcaaagttca catggaaggt tctgtaaatg gacatgaatt tgaaatagaa
     2521 ggtgaaggag aaggaaggcc ttatgaagga acccaaaccg cgaagctaaa agttactaag
     2581 ggtggcccat taccatttgc atgggatatc cttagccctc aattcatgta tgggtcaaag
     2641 gcttatgtca agcaccccgc cgacattcca gactatctaa agttatcttt tcccgaaggg
     2701 tttaagtggg agcgtgtgat gaacttcgaa gacggtggcg tggtaacagt gactcaggat
     2761 tcgtccctgc aagatggtga atttatctac aaagtcaaat taagaggaac taactttcca
     2821 tctgacggcc cggttatgca aaaaaagaca atgggctggg aggcctcctc agaacgaatg
     2881 taccctgaag atggtgcctt gaagggtgag attaaacaaa gattgaaatt gaaagatggt
     2941 ggacattatg acgctgaggt taaaacgaca tacaaagcta agaaacctgt ccagctccca
     3001 ggtgcttaca atgtaaatat aaaacttgat attacatcac ataatgaaga ttatacgata
     3061 gttgaacaat acgaaagggc tgaggggaga catagtactg gtggcatgga tgaactatac
     3121 aaataataag cggccgcgag ggccgcatca tgtaattagt tatgtcacgc ttacattcac
     3181 gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg
     3241 tccctattta tttttttata gttatgttag tattaagaac gttatttata tttcaaattt
     3301 ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg
     3361 agaaggtttt gggacgctcg aaggctttaa tttgcggcct ctagacgcct tgagcctggc
     3421 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
     3481 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
     3541 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
     3601 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag
     3661 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
     3721 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc
     3781 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
     3841 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
     3901 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtcatcga
     3961 tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat
     4021 cgccgtacca cttcaaaaca cccaagcaca gcatactaaa tttcccctct ttcttcctct
     4081 agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt
     4141 ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaaatt
     4201 tttttttgat ttttttctct ttcgatgacc tcccattgat atttaagtta ataaacggtc
     4261 ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt
     4321 gctcattaga aagaaagcat agcaatctaa tctaagtttt gggatccccc gggctgcagg
     4381 aattcgatat caagcttgga gatctaaaag aaataatgtc taaaggtgaa gaattattca
     4441 ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac aaattttctg
     4501 tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa tttatttgta
     4561 ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttcggt tatggtgttc
     4621 aatgttttgc gagataccca gatcatatga aacaacatga ctttttcaag tctgccatgc
     4681 cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac tacaagacca
     4741 gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta aaaggtattg
     4801 attttaaaga agatggtaac attttaggtc acaaattgga atacaactat aactctcaca
     4861 atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc aaaattagac
     4921 acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat actccaattg
     4981 gtgatggtcc agtcttgtta ccagacaacc attacttatc cactcaatct gccttatcca
     5041 aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact gctgctggta
     5101 ttatccatgg tatggatgaa ttgtacaaat aaagcctagg aaacaaacaa agctgtcacg
     5161 gagtggggtt ccggtctgat gagatcctca tgtgaactgc tgtcgctgag gatcgaaaca
     5221 gcaaaaagaa aaataaaaac tcgagcctcg agagggcgcg ccacttctaa ataagcgaat
     5281 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa
     5341 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg
     5401 taggtcaggt tgctttctca ggtatagtat gaggtcgctc ttattgacca cacctctacc
     5461 ggcagatccg ctagggataa cagggtaata taggtaccag cttttgttcc ctttagtgag
     5521 ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5581 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5641 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5701 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5761 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5821 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5881 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5941 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6001 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6061 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6121 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6181 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6241 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6301 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6361 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6421 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6481 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6541 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6601 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6661 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6721 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6781 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6841 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6901 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6961 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7021 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7081 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7141 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7201 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7261 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7321 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7381 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7441 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7501 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7561 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7621 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7681 ttccccgaaa agtgccacct gggtcctttt catcacgtgc tataaaaata attataattt
     7741 aaatttttta atataaatat ataaattaaa aatagaaagt aaaaaaagaa attaaagaaa
     7801 aaatagtttt tgttttccga agatgtaaaa gactctaggg ggatcgccaa caaatactac
     7861 cttttatctt gctcttcctg ctctcaggta ttaatgccga attgtttcat cttgtctgtg
     7921 tagaagacca cacacgaaaa tcctgtgatt ttacatttta cttatcgtta atcgaatgta
     7981 tatctattta atctgctttt cttgtctaat aaatatatat gtaaagtacg ctttttgttg
     8041 aaatttttta aacctttgtt tatttttttt tcttcattcc gtaactcttc taccttcttt
     8101 atttactttc taaaatccaa atacaaaaca taaaaataaa taaacacaga gtaaattccc
     8161 aaattattcc atcattaaaa gatacgaggc gcgtgtaagt tacaggcaag cgatccgtct
     8221 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     8281 gtc
//
