LOCUS       sequence-319554-        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"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        184..836
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    388..505
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     800..823
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             876..1574
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2334..2351
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2687..2706)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2688..2706)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2713..2730)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2713..2729)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2722..2744)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2870..3325
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2957..2976)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3167..3188
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3428..4102)
                     /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(4103..4383)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4404..4423)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4523..4545
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4583..4601)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4642..5145
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5182..5286
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5287..5355,5356..6147)
                     /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(5505..5524)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6318..6906
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6807..6826
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7060..7077
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag ctcgaaatta accctcacta aagggaacaa aagctggtac caattccttg
      181 aattttcaaa aattcttact ttttttttgg atggacgcaa agaagtttaa taatcatatt
      241 acatggcatt accaccatat acatatccat atctaatctt acttatatgt tgtggaaatg
      301 taaagagccc cattatctta gcctaaaaaa accttctctt tggaactttc agtaatacgc
      361 ttaactgctc attgctatat tgaagtacgg attagaagcc gccgagcggg tgacagccct
      421 ccgaaggaag actctcctcc gtgcgtcctc gtcttcaccg gtcgcgttcc tgaaacgcag
      481 atgtgcctcg cgccgcactg ctccgaacaa taaagattct acaatactag cttttatggt
      541 tatgaagagg aaaaattggc agtaacctgg ccccacaaac cttcaaatga acgaatcaaa
      601 ttaacaacca taggatgata atgcgattag ttttttagcc ttatttctgg ggtaattaat
      661 cagcgaagcg atgatttttg atctattaac agatatataa atgcaaaaac tgcataacca
      721 ctttaactaa tactttcaac attttcggtt tgtattactt cttattcaaa tgtaataaaa
      781 gtatcaacaa aaaattgtta atatacctct atactttaac gtcaaggaga aaaaaccccg
      841 gatcgaattc cctacttcat acattttcaa ttaagatgag cgagctgatt aaggagaaca
      901 tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg
      961 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     1021 gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct
     1081 tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca
     1141 catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca
     1201 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca
     1261 acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc
     1321 ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc
     1381 atctgatcgc aaacgccaag accacatata gatccaagaa acccgctaag aacctcaaga
     1441 tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga
     1501 cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg
     1561 ggcacaagct taatatggga taggctggct ccgctgctgg ttctggcgaa taggcatcca
     1621 agggcgagga gctctttact ggcgtagtac caattctcgt agagctcgat ggcgatgtaa
     1681 atggccataa gttttccgta cgcggcgagg gcgagggcga tgcaactaac ggcaagctca
     1741 ctctcaagtt tatttgtact actggcaagc tcccagtacc atggccaact ctcgtaacta
     1801 ctctgaccta tggcgtacaa tgtttttccc gctatccaga tcacatgaag caacatgatt
     1861 tttttaagtc cgcaatgcca gagggctatg tacaagagcg cactattagc tttaaggatg
     1921 atggcaccta taagactcgc gcagaggtaa agtttgaggg cgatactctc gtaaatcgca
     1981 ttgagctcaa gggcattgat tttaaggagg atggcaatat tctcggccat aagctggagt
     2041 ataatttcaa ttcccataat gtatatatta ccgcagataa gcaaaagaat ggcattaagg
     2101 cgaattttaa gattcgccat aatgtggagg atggctccgt acaactcgca gatcattatc
     2161 aacaaaatac tccaattggc gatggcccag tactcctccc agataatcat tatctctcca
     2221 ctcaatccgt gctctccaaa gatccaaatg agaagcgcga tcacatggta ctcctggagt
     2281 ttgtaactgc agcaggcatt actcatggca tggatgagct ctataagctc gagcaccacc
     2341 accaccacca ctaataatag ctcgagatct gataacaaca gtgtagatgt aacaaaatcg
     2401 actttgttcc cactgtactt ttagctcgta caaaatacaa tatacttttc atttctccgt
     2461 aaacaacatg ttttcccatg taatatcctt ttctattttt cgttccgtta ccaactttac
     2521 acatacttta tatagctatt cacttctata cactaaaaaa ctaagacaat tttaattttg
     2581 ctgcctgcca tatttcaatt tgttataaat tcctataatt tatcctatta gtagctaaaa
     2641 aaagatgaat gtgaatcgaa tcctaagaga attgagctcc aattcgccct atagtgagtc
     2701 gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2761 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2821 ccgcaccgat cgcctttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt
     2881 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2941 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3001 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3061 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3121 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3181 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3241 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3301 aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg
     3361 gtatttcaca ccgcatagat cggcaagtgc acaaacaata cttaaataaa tactactcag
     3421 taataaccta tttcttagca tttttgacga aatttgctat tttgttagag tcttttacac
     3481 catttgtctc cacacctccg cttacatcaa caccaataac gccatttaat ctaagcgcat
     3541 caccaacatt ttctggcgtc agtccaccag ctaacataaa atgtaagctt tcggggctct
     3601 cttgccttcc aacccagtca gaaatcgagt tccaatccaa aagttcacct gtcccacctg
     3661 cttctgaatc aaacaaggga ataaacgaat gaggtttctg tgaagctgca ctgagtagta
     3721 tgttgcagtc ttttggaaat acgagtcttt taataactgg caaaccgagg aactcttggt
     3781 attcttgcca cgactcatct ccatgcagtt ggacgatatc aatgccgtaa tcattgacca
     3841 gagccaaaac atcctcctta ggttgattac gaaacacgcc aaccaagtat ttcggagtgc
     3901 ctgaactatt tttatatgct tttacaagac ttgaaatttt ccttgcaata accgggtcaa
     3961 ttgttctctt tctattgggc acacatataa tacccagcaa gtcagcatcg gaatctagag
     4021 cacattctgc ggcctctgtg ctctgcaagc cgcaaacttt caccaatgga ccagaactac
     4081 ctgtgaaatt aataacagac atactccaag ctgcctttgt gtgcttaatc acgtatactc
     4141 acgtgctcaa tagtcaccaa tgccctccct cttggccctc tccttttctt ttttcgaccg
     4201 aattaattct taatcggcaa aaaaagaaaa gctccggatc aagattgtac gtaaggtgac
     4261 aagctatttt tcaataaaga atatcttcca ctactgccat ctggcgtcat aactgcaaag
     4321 tacacatata ttacgatgct gttctattaa atgcttccta tattatatat atagtaatgt
     4381 cgtgatctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4441 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4501 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4561 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4621 ataataatgg tttcttagac ggatcgcttg cctgtaactt acacgcgcct cgtatctttt
     4681 aatgatggaa taatttggga atttactctg tgtttattta tttttatgtt ttgtatttgg
     4741 attttagaaa gtaaataaag aaggtagaag agttacggaa tgaagaaaaa aaaataaaca
     4801 aaggtttaaa aaatttcaac aaaaagcgta ctttacatat atatttatta gacaagaaaa
     4861 gcagattaaa tagatataca ttcgattaac gataagtaaa atgtaaaatc acaggatttt
     4921 cgtgtgtggt cttctacaca gacaagatga aacaattcgg cattaatacc tgagagcagg
     4981 aagagcaaga taaaaggtag tatttgttgg cgatccccct agagtctttt acatcttcgg
     5041 aaaacaaaaa ctattttttc tttaatttct ttttttactt tctattttta atttatatat
     5101 ttatattaaa aaatttaaat tataattatt tttatagcac gtgatgaaaa ggacccaggt
     5161 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5221 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5281 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5341 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5401 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5461 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5521 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5581 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5641 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5701 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5761 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5821 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5881 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     5941 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6001 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6061 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6121 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6181 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6241 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6301 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6361 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6421 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6481 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6541 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6601 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6661 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6721 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6781 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6841 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6901 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6961 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     7021 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     7081 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     7141 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aat
//
