LOCUS       sequence-319615-        7193 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 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-altTAG2
FEATURES             Location/Qualifiers
     source          1..7193
                     /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(1858..1877)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1859..1877)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1884..1901)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1884..1900)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1893..1915)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2041..2496
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2128..2147)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2338..2359
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2599..3273)
                     /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(3274..3554)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3575..3594)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3694..3716
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3754..3772)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3813..4316
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4353..4457
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4458..4526,4527..5318)
                     /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(4676..4695)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5489..6077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5978..5997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6231..6248
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6365..6386
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6401..6406,6407..6424,6425..6431)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6439..6455
                     /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     6444..6466
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6463..6479
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6463..6479
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        6500..6518
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        join(6548..7193,1..7)
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    6752..6869
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     7164..7187
                     /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 atccgctggc taggctgctg 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 caattcgccc
     1861 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     1921 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     1981 agcgaagagg cccgcaccga tcgcctttcc caacagttgc gcagcctgaa tggcgaatgg
     2041 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2101 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2161 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2221 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2281 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2341 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2401 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2461 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
     2521 gcatctgtgc ggtatttcac accgcataga tcggcaagtg cacaaacaat acttaaataa
     2581 atactactca gtaataacct atttcttagc atttttgacg aaatttgcta ttttgttaga
     2641 gtcttttaca ccatttgtct ccacacctcc gcttacatca acaccaataa cgccatttaa
     2701 tctaagcgca tcaccaacat tttctggcgt cagtccacca gctaacataa aatgtaagct
     2761 ttcggggctc tcttgccttc caacccagtc agaaatcgag ttccaatcca aaagttcacc
     2821 tgtcccacct gcttctgaat caaacaaggg aataaacgaa tgaggtttct gtgaagctgc
     2881 actgagtagt atgttgcagt cttttggaaa tacgagtctt ttaataactg gcaaaccgag
     2941 gaactcttgg tattcttgcc acgactcatc tccatgcagt tggacgatat caatgccgta
     3001 atcattgacc agagccaaaa catcctcctt aggttgatta cgaaacacgc caaccaagta
     3061 tttcggagtg cctgaactat ttttatatgc ttttacaaga cttgaaattt tccttgcaat
     3121 aaccgggtca attgttctct ttctattggg cacacatata atacccagca agtcagcatc
     3181 ggaatctaga gcacattctg cggcctctgt gctctgcaag ccgcaaactt tcaccaatgg
     3241 accagaacta cctgtgaaat taataacaga catactccaa gctgcctttg tgtgcttaat
     3301 cacgtatact cacgtgctca atagtcacca atgccctccc tcttggccct ctccttttct
     3361 tttttcgacc gaattaattc ttaatcggca aaaaaagaaa agctccggat caagattgta
     3421 cgtaaggtga caagctattt ttcaataaag aatatcttcc actactgcca tctggcgtca
     3481 taactgcaaa gtacacatat attacgatgc tgttctatta aatgcttcct atattatata
     3541 tatagtaatg tcgtgatcta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3601 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3661 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3721 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     3781 ttaatgtcat gataataatg gtttcttaga cggatcgctt gcctgtaact tacacgcgcc
     3841 tcgtatcttt taatgatgga ataatttggg aatttactct gtgtttattt atttttatgt
     3901 tttgtatttg gattttagaa agtaaataaa gaaggtagaa gagttacgga atgaagaaaa
     3961 aaaaataaac aaaggtttaa aaaatttcaa caaaaagcgt actttacata tatatttatt
     4021 agacaagaaa agcagattaa atagatatac attcgattaa cgataagtaa aatgtaaaat
     4081 cacaggattt tcgtgtgtgg tcttctacac agacaagatg aaacaattcg gcattaatac
     4141 ctgagagcag gaagagcaag ataaaaggta gtatttgttg gcgatccccc tagagtcttt
     4201 tacatcttcg gaaaacaaaa actatttttt ctttaatttc tttttttact ttctattttt
     4261 aatttatata tttatattaa aaaatttaaa ttataattat ttttatagca cgtgatgaaa
     4321 aggacccagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4381 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4441 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4501 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4561 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4621 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4681 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4741 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4801 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4861 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4921 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4981 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5041 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5101 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5161 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5221 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5281 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5341 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5401 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5461 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5521 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5581 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5641 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5701 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5761 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5821 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5881 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5941 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6001 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6061 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6121 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6181 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6241 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     6301 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     6361 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     6421 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     6481 atgattacgc caagctcgaa attaaccctc actaaaggga acaaaagctg gtaccaattc
     6541 cttgaatttt caaaaattct tacttttttt ttggatggac gcaaagaagt ttaataatca
     6601 tattacatgg cattaccacc atatacatat ccatatctaa tcttacttat atgttgtgga
     6661 aatgtaaaga gccccattat cttagcctaa aaaaaccttc tctttggaac tttcagtaat
     6721 acgcttaact gctcattgct atattgaagt acggattaga agccgccgag cgggtgacag
     6781 ccctccgaag gaagactctc ctccgtgcgt cctcgtcttc accggtcgcg ttcctgaaac
     6841 gcagatgtgc ctcgcgccgc actgctccga acaataaaga ttctacaata ctagctttta
     6901 tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa atgaacgaat
     6961 caaattaaca accataggat gataatgcga ttagtttttt agccttattt ctggggtaat
     7021 taatcagcga agcgatgatt tttgatctat taacagatat ataaatgcaa aaactgcata
     7081 accactttaa ctaatacttt caacattttc ggtttgtatt acttcttatt caaatgtaat
     7141 aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag gag
//
