LOCUS       sequence-319563-        7359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual fluorescent protein reporter (BFP-GFP) for ncAA incorporation 
            (TAG construct).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7359)
  AUTHORS   Potts KA, Stieglitz JT, Lei M, Van Deventer JA
  TITLE     Reporter system architecture affects measurements of noncanonical 
            amino acid incorporation efficiency and fidelity.
  JOURNAL   Mol Syst Des Eng. 2020 Feb 1;5(2):573-588. doi: 10.1039/c9me00107g. 
            Epub 2020 Jan 23.
  PUBMED    33791108
REFERENCE   2  (bases 1 to 7359)
  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"; doi: 
            "10.1039/c9me00107g"; journalName: "Mol Syst Des Eng"; date: 
            "2020-02-1- 1"; volume: "5"; issue: "2"; pages: "573-588"
COMMENT     Sequence Label: pRS416-BXG
FEATURES             Location/Qualifiers
     source          1..7359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..118
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     413..436
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             489..1187
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1947..1964
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2334..2353)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2335..2353)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2363..2380)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2363..2379)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2372..2394)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2520..2975
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2607..2626)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2817..2838
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3106..3909)
                     /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"
     promoter        complement(3910..4125)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4149..4168)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4268..4290
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4328..4346)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4387..4890
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4927..5031
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5032..5100,5101..5892)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAVTMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5250..5269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6063..6651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6552..6571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6805..6822
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6939..6960
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6975..6980,6981..6998,6999..7005)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7013..7029
                     /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     7018..7040
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7037..7053
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7037..7053
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7072..7092
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7074..7092
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        join(7156..7359,1..449)
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
ORIGIN
        1 cggattagaa gccgccgagc gggtgacagc cctccgaagg aagactctcc tccgtgcgtc
       61 ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa
      121 caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt ggcagtaacc
      181 tggccccaca aaccttcaaa tgaacgaatc aaattaacaa ccataggatg ataatgcgat
      241 tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt ttgatctatt
      301 aacagatata taaatgcaaa aactgcataa ccactttaac taatactttc aacattttcg
      361 gtttgtatta cttcttattc aaatgtaata aaagtatcaa caaaaaattg ttaatatacc
      421 tctatacttt aacgtcaagg agaaaaaacc ccggatcgaa ttccctactt catacatttt
      481 caattaagat gagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca
      541 ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
      601 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
      661 tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg
      721 acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag
      781 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
      841 acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac
      901 tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa
      961 acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacgcc aagaccacat
     1021 atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca
     1081 gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac gaggtggcag
     1141 tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaatatg ggatccgctg
     1201 gctccgctgc tggttctggc gaataggcat ccaagggcga ggagctcttt actggcgtag
     1261 taccaattct cgtagagctc gatggcgatg taaatggcca taagttttcc gtacgcggcg
     1321 agggcgaggg cgatgcaact aacggcaagc tcactctcaa gtttatttgt actactggca
     1381 agctcccagt accatggcca actctcgtaa ctactctgac ctatggcgta caatgttttt
     1441 cccgctatcc agatcacatg aagcaacatg atttttttaa gtccgcaatg ccagagggct
     1501 atgtacaaga gcgcactatt agctttaagg atgatggcac ctataagact cgcgcagagg
     1561 taaagtttga gggcgatact ctcgtaaatc gcattgagct caagggcatt gattttaagg
     1621 aggatggcaa tattctcggc cataagctgg agtataattt caattcccat aatgtatata
     1681 ttaccgcaga taagcaaaag aatggcatta aggcgaattt taagattcgc cataatgtgg
     1741 aggatggctc cgtacaactc gcagatcatt atcaacaaaa tactccaatt ggcgatggcc
     1801 cagtactcct cccagataat cattatctct ccactcaatc cgtgctctcc aaagatccaa
     1861 atgagaagcg cgatcacatg gtactcctgg agtttgtaac tgcagcaggc attactcatg
     1921 gcatggatga gctctataag ctcgagcacc accaccacca ccactaataa tagctcgaga
     1981 tctgataaca acagtgtaga tgtaacaaaa tcgactttgt tcccactgta cttttagctc
     2041 gtacaaaata caatatactt ttcatttctc cgtaaacaac atgttttccc atgtaatatc
     2101 cttttctatt tttcgttccg ttaccaactt tacacatact ttatatagct attcacttct
     2161 atacactaaa aaactaagac aattttaatt ttgctgcctg ccatatttca atttgttata
     2221 aattcctata atttatccta ttagtagcta aaaaaagatg aatgtgaatc gaatcctaag
     2281 agaattgagc tccaattcgg tcgacctcga gggggggccc ggtacccaat tcgccctata
     2341 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2401 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2461 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     2521 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2581 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2641 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2701 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2761 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2821 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2881 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2941 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     3001 catctgtgcg gtatttcaca ccgcataggg taataactga tataattaaa ttgaagctct
     3061 aatttgtgag tttagtatac atgcatttac ttataataca gttttttagt tttgctggcc
     3121 gcatcttctc aaatatgctt cccagcctgc ttttctgtaa cgttcaccct ctaccttagc
     3181 atcccttccc tttgcaaata gtcctcttcc aacaataata atgtcagatc ctgtagagac
     3241 cacatcatcc acggttctat actgttgacc caatgcgtct cccttgtcat ctaaacccac
     3301 accgggtgtc ataatcaacc aatcgtaacc ttcatctctt ccacccatgt ctctttgagc
     3361 aataaagccg ataacaaaat ctttgtcgct cttcgcaatg tcaacagtac ccttagtata
     3421 ttctccagta gatagggagc ccttgcatga caattctgct aacatcaaaa ggcctctagg
     3481 ttcctttgtt acttcttctg ccgcctgctt caaaccgcta acaatacctg ggcccaccac
     3541 accgtgtgca ttcgtaatgt ctgcccattc tgctattctg tatacacccg cagagtactg
     3601 caatttgact gtattaccaa tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta
     3661 cttggcggat aatgccttta gcggcttaac tgtgccctcc atggaaaaat cagtcaagat
     3721 atccacatgt gtttttagta aacaaatttt gggacctaat gcttcaacta actccagtaa
     3781 ttccttggtg gtacgaacat ccaatgaagc acacaagttt gtttgctttt cgtgcatgat
     3841 attaaatagc ttggcagcaa caggactagg atgagtagca gcacgttcct tatatgtagc
     3901 tttcgacatg atttatcttc gtttcctgca ggtttttgtt ctgtgcagtt gggttaagaa
     3961 tactgggcaa tttcatgttt cttcaacact acatatgcgt atatatacca atctaagtct
     4021 gtgctccttc cttcgttctt ccttctgttc ggagattacc gaatcaaaaa aatttcaagg
     4081 aaaccgaaat caaaaaaaag aataaaaaaa aaatgatgaa ttgaaaaggt ggtatggtgc
     4141 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4201 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4261 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4321 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4381 tagacggatc gcttgcctgt aacttacacg cgcctcgtat cttttaatga tggaataatt
     4441 tgggaattta ctctgtgttt atttattttt atgttttgta tttggatttt agaaagtaaa
     4501 taaagaaggt agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt ttaaaaaatt
     4561 tcaacaaaaa gcgtacttta catatatatt tattagacaa gaaaagcaga ttaaatagat
     4621 atacattcga ttaacgataa gtaaaatgta aaatcacagg attttcgtgt gtggtcttct
     4681 acacagacaa gatgaaacaa ttcggcatta atacctgaga gcaggaagag caagataaaa
     4741 ggtagtattt gttggcgatc cccctagagt cttttacatc ttcggaaaac aaaaactatt
     4801 ttttctttaa tttctttttt tactttctat ttttaattta tatatttata ttaaaaaatt
     4861 taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac ttttcgggga
     4921 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4981 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5041 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5101 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5161 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5221 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5281 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5341 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5401 gccgtaacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5461 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5521 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5581 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5641 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5701 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5761 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5821 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5881 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5941 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6001 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6061 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6121 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6181 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6241 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6301 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6361 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6421 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6481 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6541 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6601 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6661 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6721 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6781 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     6841 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     6901 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     6961 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     7021 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
     7081 cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagagctggt
     7141 accaattcct tgaattttca aaaattctta cttttttttt ggatggacgc aaagaagttt
     7201 aataatcata ttacatggca ttaccaccat atacatatcc atatctaatc ttacttatat
     7261 gttgtggaaa tgtaaagagc cccattatct tagcctaaaa aaaccttctc tttggaactt
     7321 tcagtaatac gcttaactgc tcattgctat attgaagta
//
