LOCUS       sequence-319653-        7193 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 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-BYG
FEATURES             Location/Qualifiers
     source          1..7193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2804..3456
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3008..3125
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3420..3443
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             3496..4194
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             4954..4971
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5307..5326)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5308..5326)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5333..5350)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5333..5349)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5342..5364)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5490..5945
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5577..5596)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5787..5808
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6048..6722)
                     /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(6723..7003)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(7024..7043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7143..7165
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcgaaatta
     2761 accctcacta aagggaacaa aagctggtac caattccttg aattttcaaa aattcttact
     2821 ttttttttgg atggacgcaa agaagtttaa taatcatatt acatggcatt accaccatat
     2881 acatatccat atctaatctt acttatatgt tgtggaaatg taaagagccc cattatctta
     2941 gcctaaaaaa accttctctt tggaactttc agtaatacgc ttaactgctc attgctatat
     3001 tgaagtacgg attagaagcc gccgagcggg tgacagccct ccgaaggaag actctcctcc
     3061 gtgcgtcctc gtcttcaccg gtcgcgttcc tgaaacgcag atgtgcctcg cgccgcactg
     3121 ctccgaacaa taaagattct acaatactag cttttatggt tatgaagagg aaaaattggc
     3181 agtaacctgg ccccacaaac cttcaaatga acgaatcaaa ttaacaacca taggatgata
     3241 atgcgattag ttttttagcc ttatttctgg ggtaattaat cagcgaagcg atgatttttg
     3301 atctattaac agatatataa atgcaaaaac tgcataacca ctttaactaa tactttcaac
     3361 attttcggtt tgtattactt cttattcaaa tgtaataaaa gtatcaacaa aaaattgtta
     3421 atatacctct atactttaac gtcaaggaga aaaaaccccg gatcgaattc cctacttcat
     3481 acattttcaa ttaagatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg
     3541 gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac
     3601 gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc
     3661 gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc
     3721 atccccgact tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca
     3781 tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc
     3841 atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag
     3901 aaaacactcg gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa
     3961 ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacgccaag
     4021 accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg
     4081 gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag
     4141 gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaagct taatatggga
     4201 tccgctggct ccgctgctgg ttctggcgaa tacgcatcca agggcgagga gctctttact
     4261 ggcgtagtac caattctcgt agagctcgat ggcgatgtaa atggccataa gttttccgta
     4321 cgcggcgagg gcgagggcga tgcaactaac ggcaagctca ctctcaagtt tatttgtact
     4381 actggcaagc tcccagtacc atggccaact ctcgtaacta ctctgaccta tggcgtacaa
     4441 tgtttttccc gctatccaga tcacatgaag caacatgatt tttttaagtc cgcaatgcca
     4501 gagggctatg tacaagagcg cactattagc tttaaggatg atggcaccta taagactcgc
     4561 gcagaggtaa agtttgaggg cgatactctc gtaaatcgca ttgagctcaa gggcattgat
     4621 tttaaggagg atggcaatat tctcggccat aagctggagt ataatttcaa ttcccataat
     4681 gtatatatta ccgcagataa gcaaaagaat ggcattaagg cgaattttaa gattcgccat
     4741 aatgtggagg atggctccgt acaactcgca gatcattatc aacaaaatac tccaattggc
     4801 gatggcccag tactcctccc agataatcat tatctctcca ctcaatccgt gctctccaaa
     4861 gatccaaatg agaagcgcga tcacatggta ctcctggagt ttgtaactgc agcaggcatt
     4921 actcatggca tggatgagct ctataagctc gagcaccacc accaccacca ctaataatag
     4981 ctcgagatct gataacaaca gtgtagatgt aacaaaatcg actttgttcc cactgtactt
     5041 ttagctcgta caaaatacaa tatacttttc atttctccgt aaacaacatg ttttcccatg
     5101 taatatcctt ttctattttt cgttccgtta ccaactttac acatacttta tatagctatt
     5161 cacttctata cactaaaaaa ctaagacaat tttaattttg ctgcctgcca tatttcaatt
     5221 tgttataaat tcctataatt tatcctatta gtagctaaaa aaagatgaat gtgaatcgaa
     5281 tcctaagaga attgagctcc aattcgccct atagtgagtc gtattacaat tcactggccg
     5341 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     5401 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcctttccc
     5461 aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc
     5521 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5581 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5641 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5701 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5761 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5821 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5881 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5941 aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatagat
     6001 cggcaagtgc acaaacaata cttaaataaa tactactcag taataaccta tttcttagca
     6061 tttttgacga aatttgctat tttgttagag tcttttacac catttgtctc cacacctccg
     6121 cttacatcaa caccaataac gccatttaat ctaagcgcat caccaacatt ttctggcgtc
     6181 agtccaccag ctaacataaa atgtaagctt tcggggctct cttgccttcc aacccagtca
     6241 gaaatcgagt tccaatccaa aagttcacct gtcccacctg cttctgaatc aaacaaggga
     6301 ataaacgaat gaggtttctg tgaagctgca ctgagtagta tgttgcagtc ttttggaaat
     6361 acgagtcttt taataactgg caaaccgagg aactcttggt attcttgcca cgactcatct
     6421 ccatgcagtt ggacgatatc aatgccgtaa tcattgacca gagccaaaac atcctcctta
     6481 ggttgattac gaaacacgcc aaccaagtat ttcggagtgc ctgaactatt tttatatgct
     6541 tttacaagac ttgaaatttt ccttgcaata accgggtcaa ttgttctctt tctattgggc
     6601 acacatataa tacccagcaa gtcagcatcg gaatctagag cacattctgc ggcctctgtg
     6661 ctctgcaagc cgcaaacttt caccaatgga ccagaactac ctgtgaaatt aataacagac
     6721 atactccaag ctgcctttgt gtgcttaatc acgtatactc acgtgctcaa tagtcaccaa
     6781 tgccctccct cttggccctc tccttttctt ttttcgaccg aattaattct taatcggcaa
     6841 aaaaagaaaa gctccggatc aagattgtac gtaaggtgac aagctatttt tcaataaaga
     6901 atatcttcca ctactgccat ctggcgtcat aactgcaaag tacacatata ttacgatgct
     6961 gttctattaa atgcttccta tattatatat atagtaatgt cgtgatctat ggtgcactct
     7021 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     7081 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     7141 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cga
//
