LOCUS       sequence-319621-        7359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual fluorescent protein reporter (BFP-GFP) for ncAA incorporation 
            (wild-type 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-BYG
FEATURES             Location/Qualifiers
     source          1..7359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     295..318
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             371..1069
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1829..1846
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2216..2235)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2217..2235)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2245..2262)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2245..2261)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2254..2276)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2402..2857
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2489..2508)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2699..2720
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2988..3791)
                     /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(3792..4007)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4031..4050)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4150..4172
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4210..4228)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4269..4772
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4809..4913
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4914..4982,4983..5774)
                     /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(5132..5151)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5945..6533
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6434..6453
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6687..6704
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6821..6842
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6857..6862,6863..6880,6881..6887)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6895..6911
                     /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     6900..6922
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6919..6935
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6919..6935
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6954..6974
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6956..6974
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        join(7038..7359,1..331)
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    7242..7359
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
ORIGIN
        1 aacaataaag attctacaat actagctttt atggttatga agaggaaaaa ttggcagtaa
       61 cctggcccca caaaccttca aatgaacgaa tcaaattaac aaccatagga tgataatgcg
      121 attagttttt tagccttatt tctggggtaa ttaatcagcg aagcgatgat ttttgatcta
      181 ttaacagata tataaatgca aaaactgcat aaccacttta actaatactt tcaacatttt
      241 cggtttgtat tacttcttat tcaaatgtaa taaaagtatc aacaaaaaat tgttaatata
      301 cctctatact ttaacgtcaa ggagaaaaaa ccccggatcg aattccctac ttcatacatt
      361 ttcaattaag atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
      421 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
      481 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
      541 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
      601 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
      661 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
      721 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
      781 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
      841 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaacg ccaagaccac
      901 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
      961 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     1021 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaata tgggatccgc
     1081 tggctccgct gctggttctg gcgaatacgc atccaagggc gaggagctct ttactggcgt
     1141 agtaccaatt ctcgtagagc tcgatggcga tgtaaatggc cataagtttt ccgtacgcgg
     1201 cgagggcgag ggcgatgcaa ctaacggcaa gctcactctc aagtttattt gtactactgg
     1261 caagctccca gtaccatggc caactctcgt aactactctg acctatggcg tacaatgttt
     1321 ttcccgctat ccagatcaca tgaagcaaca tgattttttt aagtccgcaa tgccagaggg
     1381 ctatgtacaa gagcgcacta ttagctttaa ggatgatggc acctataaga ctcgcgcaga
     1441 ggtaaagttt gagggcgata ctctcgtaaa tcgcattgag ctcaagggca ttgattttaa
     1501 ggaggatggc aatattctcg gccataagct ggagtataat ttcaattccc ataatgtata
     1561 tattaccgca gataagcaaa agaatggcat taaggcgaat tttaagattc gccataatgt
     1621 ggaggatggc tccgtacaac tcgcagatca ttatcaacaa aatactccaa ttggcgatgg
     1681 cccagtactc ctcccagata atcattatct ctccactcaa tccgtgctct ccaaagatcc
     1741 aaatgagaag cgcgatcaca tggtactcct ggagtttgta actgcagcag gcattactca
     1801 tggcatggat gagctctata agctcgagca ccaccaccac caccactaat aatagctcga
     1861 gatctgataa caacagtgta gatgtaacaa aatcgacttt gttcccactg tacttttagc
     1921 tcgtacaaaa tacaatatac ttttcatttc tccgtaaaca acatgttttc ccatgtaata
     1981 tccttttcta tttttcgttc cgttaccaac tttacacata ctttatatag ctattcactt
     2041 ctatacacta aaaaactaag acaattttaa ttttgctgcc tgccatattt caatttgtta
     2101 taaattccta taatttatcc tattagtagc taaaaaaaga tgaatgtgaa tcgaatccta
     2161 agagaattga gctccaattc ggtcgacctc gagggggggc ccggtaccca attcgcccta
     2221 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     2281 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     2341 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     2401 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2461 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2521 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2581 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2641 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2701 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2761 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2821 aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta
     2881 cgcatctgtg cggtatttca caccgcatag ggtaataact gatataatta aattgaagct
     2941 ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta gttttgctgg
     3001 ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc ctctacctta
     3061 gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga tcctgtagag
     3121 accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc atctaaaccc
     3181 acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat gtctctttga
     3241 gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt acccttagta
     3301 tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa aaggcctcta
     3361 ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc
     3421 acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc cgcagagtac
     3481 tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag taaaaaattg
     3541 tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa atcagtcaag
     3601 atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac taactccagt
     3661 aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg
     3721 atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc cttatatgta
     3781 gctttcgaca tgatttatct tcgtttcctg caggtttttg ttctgtgcag ttgggttaag
     3841 aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac caatctaagt
     3901 ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa
     3961 ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag gtggtatggt
     4021 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     4081 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4141 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4201 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4261 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
     4321 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
     4381 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
     4441 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
     4501 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
     4561 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
     4621 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
     4681 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
     4741 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
     4801 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4861 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4921 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4981 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5041 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5101 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5161 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5221 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5281 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5341 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5401 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5461 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5521 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5581 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5641 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5701 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5761 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5821 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5881 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5941 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6001 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6061 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6121 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6181 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6241 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6301 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6361 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6421 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6481 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6541 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6601 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     6661 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     6721 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     6781 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     6841 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     6901 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     6961 accctcacta aagggaacaa aagctggagc tccaccgcgg tggcggccgc tctagagctg
     7021 gtaccaattc cttgaatttt caaaaattct tacttttttt ttggatggac gcaaagaagt
     7081 ttaataatca tattacatgg cattaccacc atatacatat ccatatctaa tcttacttat
     7141 atgttgtgga aatgtaaaga gccccattat cttagcctaa aaaaaccttc tctttggaac
     7201 tttcagtaat acgcttaact gctcattgct atattgaagt acggattaga agccgccgag
     7261 cgggtgacag ccctccgaag gaagactctc ctccgtgcgt cctcgtcttc accggtcgcg
     7321 ttcctgaaac gcagatgtgc ctcgcgccgc actgctccg
//
