LOCUS       sequence-310738-        7193 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual fluorescent protein reporter (BFP-GFP) for ncAA incorporation 
            (2 TAG construct).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7193)
  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 7193)
  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: pCTCON2-BXG-2TAG
FEATURES             Location/Qualifiers
     source          1..7193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(170..189)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     289..311
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(349..367)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    408..911
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        948..1052
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1053..1121,1122..1913)
                     /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(1271..1290)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2084..2672
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2573..2592
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2826..2843
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2960..2981
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2996..3001,3002..3019,3020..3026)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3034..3050
                     /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     3039..3061
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3058..3074
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3058..3074
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3095..3113
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3143..3795
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3347..3464
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3759..3782
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             3835..4533
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             5293..5310
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5646..5665)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5647..5665)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5672..5689)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5672..5688)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5681..5703)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5829..6284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5916..5935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6126..6147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6387..7061)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(join(7062..7193,1..149))
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
ORIGIN
        1 cggatcaaga ttgtacgtaa ggtgacaagc tatttttcaa taaagaatat cttccactac
       61 tgccatctgg cgtcataact gcaaagtaca catatattac gatgctgttc tattaaatgc
      121 ttcctatatt atatatatag taatgtcgtg atctatggtg cactctcagt acaatctgct
      181 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
      241 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
      301 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
      361 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacggat cgcttgcctg
      421 taacttacac gcgcctcgta tcttttaatg atggaataat ttgggaattt actctgtgtt
      481 tatttatttt tatgttttgt atttggattt tagaaagtaa ataaagaagg tagaagagtt
      541 acggaatgaa gaaaaaaaaa taaacaaagg tttaaaaaat ttcaacaaaa agcgtacttt
      601 acatatatat ttattagaca agaaaagcag attaaataga tatacattcg attaacgata
      661 agtaaaatgt aaaatcacag gattttcgtg tgtggtcttc tacacagaca agatgaaaca
      721 attcggcatt aatacctgag agcaggaaga gcaagataaa aggtagtatt tgttggcgat
      781 ccccctagag tcttttacat cttcggaaaa caaaaactat tttttcttta atttcttttt
      841 ttactttcta tttttaattt atatatttat attaaaaaat ttaaattata attattttta
      901 tagcacgtga tgaaaaggac ccaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      961 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1021 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1081 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1141 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1201 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1261 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1321 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1381 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1441 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1501 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1561 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     1621 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1681 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1741 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     1801 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1861 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1921 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1981 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2041 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2101 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2161 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2221 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2281 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2341 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2401 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2461 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2521 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2581 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2641 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2701 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2761 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     2821 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     2881 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     2941 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     3001 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     3061 gaaacagcta tgaccatgat tacgccaagc tcgaaattaa ccctcactaa agggaacaaa
     3121 agctggtacc aattccttga attttcaaaa attcttactt tttttttgga tggacgcaaa
     3181 gaagtttaat aatcatatta catggcatta ccaccatata catatccata tctaatctta
     3241 cttatatgtt gtggaaatgt aaagagcccc attatcttag cctaaaaaaa ccttctcttt
     3301 ggaactttca gtaatacgct taactgctca ttgctatatt gaagtacgga ttagaagccg
     3361 ccgagcgggt gacagccctc cgaaggaaga ctctcctccg tgcgtcctcg tcttcaccgg
     3421 tcgcgttcct gaaacgcaga tgtgcctcgc gccgcactgc tccgaacaat aaagattcta
     3481 caatactagc ttttatggtt atgaagagga aaaattggca gtaacctggc cccacaaacc
     3541 ttcaaatgaa cgaatcaaat taacaaccat aggatgataa tgcgattagt tttttagcct
     3601 tatttctggg gtaattaatc agcgaagcga tgatttttga tctattaaca gatatataaa
     3661 tgcaaaaact gcataaccac tttaactaat actttcaaca ttttcggttt gtattacttc
     3721 ttattcaaat gtaataaaag tatcaacaaa aaattgttaa tatacctcta tactttaacg
     3781 tcaaggagaa aaaaccccgg atcgaattcc ctacttcata cattttcaat taagatgagc
     3841 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     3901 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     3961 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     4021 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     4081 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     4141 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     4201 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     4261 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     4321 aagctcgtgg gcgggagcca tctgatcgca aacgccaaga ccacatatag atccaagaaa
     4381 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     4441 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     4501 gacctcccta gcaaactggg gcacaagctt aatatgggat aggctggctc cgctgctggt
     4561 tctggcgaat aggcatccaa gggcgaggag ctctttactg gcgtagtacc aattctcgta
     4621 gagctcgatg gcgatgtaaa tggccataag ttttccgtac gcggcgaggg cgagggcgat
     4681 gcaactaacg gcaagctcac tctcaagttt atttgtacta ctggcaagct cccagtacca
     4741 tggccaactc tcgtaactac tctgacctat ggcgtacaat gtttttcccg ctatccagat
     4801 cacatgaagc aacatgattt ttttaagtcc gcaatgccag agggctatgt acaagagcgc
     4861 actattagct ttaaggatga tggcacctat aagactcgcg cagaggtaaa gtttgagggc
     4921 gatactctcg taaatcgcat tgagctcaag ggcattgatt ttaaggagga tggcaatatt
     4981 ctcggccata agctggagta taatttcaat tcccataatg tatatattac cgcagataag
     5041 caaaagaatg gcattaaggc gaattttaag attcgccata atgtggagga tggctccgta
     5101 caactcgcag atcattatca acaaaatact ccaattggcg atggcccagt actcctccca
     5161 gataatcatt atctctccac tcaatccgtg ctctccaaag atccaaatga gaagcgcgat
     5221 cacatggtac tcctggagtt tgtaactgca gcaggcatta ctcatggcat ggatgagctc
     5281 tataagctcg agcaccacca ccaccaccac taataatagc tcgagatctg ataacaacag
     5341 tgtagatgta acaaaatcga ctttgttccc actgtacttt tagctcgtac aaaatacaat
     5401 atacttttca tttctccgta aacaacatgt tttcccatgt aatatccttt tctatttttc
     5461 gttccgttac caactttaca catactttat atagctattc acttctatac actaaaaaac
     5521 taagacaatt ttaattttgc tgcctgccat atttcaattt gttataaatt cctataattt
     5581 atcctattag tagctaaaaa aagatgaatg tgaatcgaat cctaagagaa ttgagctcca
     5641 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
     5701 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     5761 ggcgtaatag cgaagaggcc cgcaccgatc gcctttccca acagttgcgc agcctgaatg
     5821 gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5881 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5941 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     6001 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     6061 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     6121 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     6181 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     6241 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt
     6301 ctccttacgc atctgtgcgg tatttcacac cgcatagatc ggcaagtgca caaacaatac
     6361 ttaaataaat actactcagt aataacctat ttcttagcat ttttgacgaa atttgctatt
     6421 ttgttagagt cttttacacc atttgtctcc acacctccgc ttacatcaac accaataacg
     6481 ccatttaatc taagcgcatc accaacattt tctggcgtca gtccaccagc taacataaaa
     6541 tgtaagcttt cggggctctc ttgccttcca acccagtcag aaatcgagtt ccaatccaaa
     6601 agttcacctg tcccacctgc ttctgaatca aacaagggaa taaacgaatg aggtttctgt
     6661 gaagctgcac tgagtagtat gttgcagtct tttggaaata cgagtctttt aataactggc
     6721 aaaccgagga actcttggta ttcttgccac gactcatctc catgcagttg gacgatatca
     6781 atgccgtaat cattgaccag agccaaaaca tcctccttag gttgattacg aaacacgcca
     6841 accaagtatt tcggagtgcc tgaactattt ttatatgctt ttacaagact tgaaattttc
     6901 cttgcaataa ccgggtcaat tgttctcttt ctattgggca cacatataat acccagcaag
     6961 tcagcatcgg aatctagagc acattctgcg gcctctgtgc tctgcaagcc gcaaactttc
     7021 accaatggac cagaactacc tgtgaaatta ataacagaca tactccaagc tgcctttgtg
     7081 tgcttaatca cgtatactca cgtgctcaat agtcaccaat gccctccctc ttggccctct
     7141 ccttttcttt tttcgaccga attaattctt aatcggcaaa aaaagaaaag ctc
//
