LOCUS       sequence-319559-        7193 bp    DNA     circular SYN 15-APR-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 Apr 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-altTAG4
FEATURES             Location/Qualifiers
     source          1..7193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..118
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     413..436
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             489..1187
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1947..1964
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2300..2319)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2301..2319)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2326..2343)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2326..2342)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2335..2357)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2483..2938
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2570..2589)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2780..2801
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3041..3715)
                     /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(3716..3996)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4017..4036)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4136..4158
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4196..4214)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4255..4758
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4795..4899
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4900..4968,4969..5760)
                     /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(5118..5137)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5931..6519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6420..6439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6673..6690
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6807..6828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6843..6848,6849..6866,6867..6873)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6881..6897
                     /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     6886..6908
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6905..6921
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6905..6921
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        6942..6960
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        join(6990..7193,1..449)
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
ORIGIN
        1 cggattagaa gccgccgagc gggtgacagc cctccgaagg aagactctcc tccgtgcgtc
       61 ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa
      121 caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt ggcagtaacc
      181 tggccccaca aaccttcaaa tgaacgaatc aaattaacaa ccataggatg ataatgcgat
      241 tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt ttgatctatt
      301 aacagatata taaatgcaaa aactgcataa ccactttaac taatactttc aacattttcg
      361 gtttgtatta cttcttattc aaatgtaata aaagtatcaa caaaaaattg ttaatatacc
      421 tctatacttt aacgtcaagg agaaaaaacc ccggatcgaa ttccctactt catacatttt
      481 caattaagat gagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca
      541 ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
      601 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
      661 tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg
      721 acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag
      781 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
      841 acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac
      901 tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa
      961 acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacgcc aagaccacat
     1021 atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca
     1081 gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac gaggtggcag
     1141 tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaatatg ggatccgctg
     1201 gctccgctgc tggttagggc gaatacgcat ccaagggcga ggagctcttt actggcgtag
     1261 taccaattct cgtagagctc gatggcgatg taaatggcca taagttttcc gtacgcggcg
     1321 agggcgaggg cgatgcaact aacggcaagc tcactctcaa gtttatttgt actactggca
     1381 agctcccagt accatggcca actctcgtaa ctactctgac ctatggcgta caatgttttt
     1441 cccgctatcc agatcacatg aagcaacatg atttttttaa gtccgcaatg ccagagggct
     1501 atgtacaaga gcgcactatt agctttaagg atgatggcac ctataagact cgcgcagagg
     1561 taaagtttga gggcgatact ctcgtaaatc gcattgagct caagggcatt gattttaagg
     1621 aggatggcaa tattctcggc cataagctgg agtataattt caattcccat aatgtatata
     1681 ttaccgcaga taagcaaaag aatggcatta aggcgaattt taagattcgc cataatgtgg
     1741 aggatggctc cgtacaactc gcagatcatt atcaacaaaa tactccaatt ggcgatggcc
     1801 cagtactcct cccagataat cattatctct ccactcaatc cgtgctctcc aaagatccaa
     1861 atgagaagcg cgatcacatg gtactcctgg agtttgtaac tgcagcaggc attactcatg
     1921 gcatggatga gctctataag ctcgagcacc accaccacca ccactaataa tagctcgaga
     1981 tctgataaca acagtgtaga tgtaacaaaa tcgactttgt tcccactgta cttttagctc
     2041 gtacaaaata caatatactt ttcatttctc cgtaaacaac atgttttccc atgtaatatc
     2101 cttttctatt tttcgttccg ttaccaactt tacacatact ttatatagct attcacttct
     2161 atacactaaa aaactaagac aattttaatt ttgctgcctg ccatatttca atttgttata
     2221 aattcctata atttatccta ttagtagcta aaaaaagatg aatgtgaatc gaatcctaag
     2281 agaattgagc tccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt
     2341 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     2401 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccttt cccaacagtt
     2461 gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2521 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2581 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2641 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2701 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2761 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2821 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2881 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc
     2941 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata gatcggcaag
     3001 tgcacaaaca atacttaaat aaatactact cagtaataac ctatttctta gcatttttga
     3061 cgaaatttgc tattttgtta gagtctttta caccatttgt ctccacacct ccgcttacat
     3121 caacaccaat aacgccattt aatctaagcg catcaccaac attttctggc gtcagtccac
     3181 cagctaacat aaaatgtaag ctttcggggc tctcttgcct tccaacccag tcagaaatcg
     3241 agttccaatc caaaagttca cctgtcccac ctgcttctga atcaaacaag ggaataaacg
     3301 aatgaggttt ctgtgaagct gcactgagta gtatgttgca gtcttttgga aatacgagtc
     3361 ttttaataac tggcaaaccg aggaactctt ggtattcttg ccacgactca tctccatgca
     3421 gttggacgat atcaatgccg taatcattga ccagagccaa aacatcctcc ttaggttgat
     3481 tacgaaacac gccaaccaag tatttcggag tgcctgaact atttttatat gcttttacaa
     3541 gacttgaaat tttccttgca ataaccgggt caattgttct ctttctattg ggcacacata
     3601 taatacccag caagtcagca tcggaatcta gagcacattc tgcggcctct gtgctctgca
     3661 agccgcaaac tttcaccaat ggaccagaac tacctgtgaa attaataaca gacatactcc
     3721 aagctgcctt tgtgtgctta atcacgtata ctcacgtgct caatagtcac caatgccctc
     3781 cctcttggcc ctctcctttt cttttttcga ccgaattaat tcttaatcgg caaaaaaaga
     3841 aaagctccgg atcaagattg tacgtaaggt gacaagctat ttttcaataa agaatatctt
     3901 ccactactgc catctggcgt cataactgca aagtacacat atattacgat gctgttctat
     3961 taaatgcttc ctatattata tatatagtaa tgtcgtgatc tatggtgcac tctcagtaca
     4021 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     4081 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4141 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     4201 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacggatcgc
     4261 ttgcctgtaa cttacacgcg cctcgtatct tttaatgatg gaataatttg ggaatttact
     4321 ctgtgtttat ttatttttat gttttgtatt tggattttag aaagtaaata aagaaggtag
     4381 aagagttacg gaatgaagaa aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc
     4441 gtactttaca tatatattta ttagacaaga aaagcagatt aaatagatat acattcgatt
     4501 aacgataagt aaaatgtaaa atcacaggat tttcgtgtgt ggtcttctac acagacaaga
     4561 tgaaacaatt cggcattaat acctgagagc aggaagagca agataaaagg tagtatttgt
     4621 tggcgatccc cctagagtct tttacatctt cggaaaacaa aaactatttt ttctttaatt
     4681 tcttttttta ctttctattt ttaatttata tatttatatt aaaaaattta aattataatt
     4741 atttttatag cacgtgatga aaaggaccca ggtggcactt ttcggggaaa tgtgcgcgga
     4801 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4861 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4921 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4981 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5041 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5101 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5161 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5221 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5281 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5341 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5401 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5461 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5521 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5581 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5641 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5701 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5761 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5821 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5881 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5941 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6001 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6061 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6121 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6181 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6241 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6301 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6361 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6421 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6481 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6541 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6601 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     6661 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     6721 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     6781 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     6841 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     6901 cacacaggaa acagctatga ccatgattac gccaagctcg aaattaaccc tcactaaagg
     6961 gaacaaaagc tggtaccaat tccttgaatt ttcaaaaatt cttacttttt ttttggatgg
     7021 acgcaaagaa gtttaataat catattacat ggcattacca ccatatacat atccatatct
     7081 aatcttactt atatgttgtg gaaatgtaaa gagccccatt atcttagcct aaaaaaacct
     7141 tctctttgga actttcagta atacgcttaa ctgctcattg ctatattgaa gta
//
