LOCUS       sequence-319885-        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-altTAG
FEATURES             Location/Qualifiers
     source          1..7359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             47..745
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1505..1522
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1892..1911)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1893..1911)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1921..1938)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1921..1937)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1930..1952)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2078..2533
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2165..2184)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2375..2396
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2664..3467)
                     /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(3468..3683)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3707..3726)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3826..3848
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3886..3904)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3945..4448
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4485..4589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4590..4658,4659..5450)
                     /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(4808..4827)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5621..6209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6110..6129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6363..6380
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6497..6518
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6533..6538,6539..6556,6557..6563)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6571..6587
                     /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     6576..6598
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6595..6611
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6595..6611
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6630..6650
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6632..6650
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        join(6714..7359,1..7)
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    6918..7035
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     7330..7353
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
ORIGIN
        1 aaaaaacccc ggatcgaatt ccctacttca tacattttca attaagatga gcgagctgat
       61 taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc atcacttcaa
      121 gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt
      181 ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct tcctctacgg
      241 cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc agtccttccc
      301 tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac
      361 ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa
      421 cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccttcaccga
      481 gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc tgaagctcgt
      541 gggcgggagc catctgatcg caaacgccaa gaccacatat agatccaaga aacccgctaa
      601 gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc
      661 caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact gcgacctccc
      721 tagcaaactg gggcacaagc ttaatatggg ataggctggc tccgctgctg gttctggcga
      781 atacgcatcc aagggcgagg agctctttac tggcgtagta ccaattctcg tagagctcga
      841 tggcgatgta aatggccata agttttccgt acgcggcgag ggcgagggcg atgcaactaa
      901 cggcaagctc actctcaagt ttatttgtac tactggcaag ctcccagtac catggccaac
      961 tctcgtaact actctgacct atggcgtaca atgtttttcc cgctatccag atcacatgaa
     1021 gcaacatgat ttttttaagt ccgcaatgcc agagggctat gtacaagagc gcactattag
     1081 ctttaaggat gatggcacct ataagactcg cgcagaggta aagtttgagg gcgatactct
     1141 cgtaaatcgc attgagctca agggcattga ttttaaggag gatggcaata ttctcggcca
     1201 taagctggag tataatttca attcccataa tgtatatatt accgcagata agcaaaagaa
     1261 tggcattaag gcgaatttta agattcgcca taatgtggag gatggctccg tacaactcgc
     1321 agatcattat caacaaaata ctccaattgg cgatggccca gtactcctcc cagataatca
     1381 ttatctctcc actcaatccg tgctctccaa agatccaaat gagaagcgcg atcacatggt
     1441 actcctggag tttgtaactg cagcaggcat tactcatggc atggatgagc tctataagct
     1501 cgagcaccac caccaccacc actaataata gctcgagatc tgataacaac agtgtagatg
     1561 taacaaaatc gactttgttc ccactgtact tttagctcgt acaaaataca atatactttt
     1621 catttctccg taaacaacat gttttcccat gtaatatcct tttctatttt tcgttccgtt
     1681 accaacttta cacatacttt atatagctat tcacttctat acactaaaaa actaagacaa
     1741 ttttaatttt gctgcctgcc atatttcaat ttgttataaa ttcctataat ttatcctatt
     1801 agtagctaaa aaaagatgaa tgtgaatcga atcctaagag aattgagctc caattcggtc
     1861 gacctcgagg gggggcccgg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     1921 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     1981 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     2041 cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag cggcgcatta
     2101 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2161 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2221 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2281 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2341 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2401 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2461 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2521 acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2581 gcatagggta ataactgata taattaaatt gaagctctaa tttgtgagtt tagtatacat
     2641 gcatttactt ataatacagt tttttagttt tgctggccgc atcttctcaa atatgcttcc
     2701 cagcctgctt ttctgtaacg ttcaccctct accttagcat cccttccctt tgcaaatagt
     2761 cctcttccaa caataataat gtcagatcct gtagagacca catcatccac ggttctatac
     2821 tgttgaccca atgcgtctcc cttgtcatct aaacccacac cgggtgtcat aatcaaccaa
     2881 tcgtaacctt catctcttcc acccatgtct ctttgagcaa taaagccgat aacaaaatct
     2941 ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga tagggagccc
     3001 ttgcatgaca attctgctaa catcaaaagg cctctaggtt cctttgttac ttcttctgcc
     3061 gcctgcttca aaccgctaac aatacctggg cccaccacac cgtgtgcatt cgtaatgtct
     3121 gcccattctg ctattctgta tacacccgca gagtactgca atttgactgt attaccaatg
     3181 tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact tggcggataa tgcctttagc
     3241 ggcttaactg tgccctccat ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa
     3301 caaattttgg gacctaatgc ttcaactaac tccagtaatt ccttggtggt acgaacatcc
     3361 aatgaagcac acaagtttgt ttgcttttcg tgcatgatat taaatagctt ggcagcaaca
     3421 ggactaggat gagtagcagc acgttcctta tatgtagctt tcgacatgat ttatcttcgt
     3481 ttcctgcagg tttttgttct gtgcagttgg gttaagaata ctgggcaatt tcatgtttct
     3541 tcaacactac atatgcgtat atataccaat ctaagtctgt gctccttcct tcgttcttcc
     3601 ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa accgaaatca aaaaaaagaa
     3661 taaaaaaaaa atgatgaatt gaaaaggtgg tatggtgcac tctcagtaca atctgctctg
     3721 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3781 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3841 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     3901 tatttttata ggttaatgtc atgataataa tggtttctta gacggatcgc ttgcctgtaa
     3961 cttacacgcg cctcgtatct tttaatgatg gaataatttg ggaatttact ctgtgtttat
     4021 ttatttttat gttttgtatt tggattttag aaagtaaata aagaaggtag aagagttacg
     4081 gaatgaagaa aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc gtactttaca
     4141 tatatattta ttagacaaga aaagcagatt aaatagatat acattcgatt aacgataagt
     4201 aaaatgtaaa atcacaggat tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt
     4261 cggcattaat acctgagagc aggaagagca agataaaagg tagtatttgt tggcgatccc
     4321 cctagagtct tttacatctt cggaaaacaa aaactatttt ttctttaatt tcttttttta
     4381 ctttctattt ttaatttata tatttatatt aaaaaattta aattataatt atttttatag
     4441 cacgtgatga aaaggaccca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4501 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4561 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4621 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4681 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4741 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4801 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4861 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4921 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4981 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5041 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5101 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5161 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5221 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5281 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5341 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5401 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5461 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5521 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5581 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5641 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5701 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5761 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5821 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5881 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5941 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6001 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6061 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6121 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6181 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6241 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6301 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6361 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     6421 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     6481 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     6541 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     6601 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
     6661 tggagctcca ccgcggtggc ggccgctcta gagctggtac caattccttg aattttcaaa
     6721 aattcttact ttttttttgg atggacgcaa agaagtttaa taatcatatt acatggcatt
     6781 accaccatat acatatccat atctaatctt acttatatgt tgtggaaatg taaagagccc
     6841 cattatctta gcctaaaaaa accttctctt tggaactttc agtaatacgc ttaactgctc
     6901 attgctatat tgaagtacgg attagaagcc gccgagcggg tgacagccct ccgaaggaag
     6961 actctcctcc gtgcgtcctc gtcttcaccg gtcgcgttcc tgaaacgcag atgtgcctcg
     7021 cgccgcactg ctccgaacaa taaagattct acaatactag cttttatggt tatgaagagg
     7081 aaaaattggc agtaacctgg ccccacaaac cttcaaatga acgaatcaaa ttaacaacca
     7141 taggatgata atgcgattag ttttttagcc ttatttctgg ggtaattaat cagcgaagcg
     7201 atgatttttg atctattaac agatatataa atgcaaaaac tgcataacca ctttaactaa
     7261 tactttcaac attttcggtt tgtattactt cttattcaaa tgtaataaaa gtatcaacaa
     7321 aaaattgtta atatacctct atactttaac gtcaaggag
//
