LOCUS       sequence-298191-        5659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal gfp and orthogonal rfp genes in bacterial 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5659)
  AUTHORS   Aleksashin NA, Szal T, d'Aquino AE, Jewett MC, Vazquez-Laslop N, 
            Mankin AS
  TITLE     A fully orthogonal system for protein synthesis in bacterial cells.
  JOURNAL   Nat Commun. 2020 Apr 20;11(1):1858. doi: 10.1038/s41467-020-15756-1.
  PUBMED    32313034
REFERENCE   2  (bases 1 to 5659)
  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"; journalName: "Nat 
            Commun."; date: "2020-04-20"; volume: "11(1):1858. doi"; pages: " 
            10.1038/s41467-020-15756-1"
COMMENT     Sequence Label: poRFP-oGFP
FEATURES             Location/Qualifiers
     source          1..5659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    111..134
                     /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)."
     CDS             814..861
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             862..885
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(958..975)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1128..1214
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1306..1333
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        complement(join(1779..1784,1785..1801,1802..1807))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      1919..2464
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     2365..2384
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2581..2598
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(3074..3088,3089..3094,3095..3111,3112..3118)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3094..3110
                     /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)."
     protein_bind    3126..3142
                     /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)."
     CDS             3180..3857
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      3921..4015
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             4650..5441
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 ccgcattaaa atctagcgag gatccgaggc gcgcccagag attttgagac acaacgtggc
       61 tttccatcaa aaaaatattg acaacataaa aaactttgtg ttatacttgt ggaattgtga
      121 gcggataaca attctatatc tgttattttt tccaaccaca gatctatgag caaaggtgaa
      181 gaactgttta ccggcgttgt gccgattctg gtggaactgg atggcgatgt gaacggtcac
      241 aaattcagcg tgcgtggtga aggtgaaggc gatgccacga ttggcaaact gacgctgaaa
      301 tttatctgca ccaccggcaa actgccggtg ccgtggccga cgctggtgac caccctgacc
      361 tatggcgttc agtgttttag tcgctatccg gatcacatga aacgtcacga tttctttaaa
      421 tctgcaatgc cggaaggcta tgtgcaggaa cgtacgatta gctttaaaga tgatggcaaa
      481 tataaaacgc gcgccgttgt gaaatttgaa ggcgataccc tggtgaaccg cattgaactg
      541 aaaggcacgg attttaaaga agatggcaat atcctgggcc ataaactgga atacaacttt
      601 aatagccata atgtttatat tacggcggat aaacagaaaa atggcatcaa agcgaatttt
      661 accgttcgcc ataacgttga agatggcagt gtgcagctgg cagatcatta tcagcagaat
      721 accccgattg gtgatggtcc ggtgctgctg ccggataatc attatctgag cacgcagacc
      781 gttctgtcta aagatccgaa cgaaaaaggc acgcgggacc acatggttct gcacgaatat
      841 gtgaatgcgg caggtattac gtggagccat ccgcagttcg aaaaataagt cgaccggctg
      901 ctaacaaagc ccgcggccgc tgaagatcga tctcgacgag tgagagaaga ttttcagcct
      961 gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag
     1021 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
     1081 tggtagtgtg gggtcacccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
     1141 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     1201 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     1261 ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga
     1321 cggatggcct ttttgcgttt ctacactcga ggatctcttt cctttccatc aaaaaaatat
     1381 tgatgaaatg agctgtgggg cgttatttag gttttttctt ctttcgaaaa aatctttctt
     1441 tatgaagtta aaagctatgt attcaatagc atattttgaa tatggacata gaatagtgct
     1501 tatcactatt gcatatagca tcttatctga cacaaggaaa taataccctt cgctgttttt
     1561 tgttataagg tatatatata taagtgtgca gtacaggcca aataaaatat tttttatgta
     1621 gtatcttaaa tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc
     1681 atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc
     1741 ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag
     1801 ctgtcaaaca tgagaattac aacttatatc gtatggggct gacttcaggt gctacatttg
     1861 aagagataaa ttgcactgaa atctagaaat attttatctg attaataaga tgatcttctt
     1921 gagatcgttt tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc gccttgcagg
     1981 gcggtttttc gaaggttctc tgagctacca actctttgaa ccgaggtaac tggcttggag
     2041 gagcgcagtc accaaaactt gtcctttcag tttagcctta accggcgcat gacttcaaga
     2101 ctaactcctc taaatcaatt accagtggct gctgccagtg gtgcttttgc atgtctttcc
     2161 gggttggact caagacgata gttaccggat aaggcgcagc ggtcggactg aacggggggt
     2221 tcgtgcatac agtccagctt ggagcgaact gcctacccgg aactgagtgt caggcgtgga
     2281 atgagacaaa cgcggccata acagcggaat gacaccggta aaccgaaagg caggaacagg
     2341 agagcgcacg agggagccgc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2401 tcgccaccac tgatttgagc gtcagatttc gtgatgcttg tcaggggggc ggagcctatg
     2461 gaaaaacggc tttgccgcgg ccctctcact tccctgttaa gtatcttcct ggcatcttcc
     2521 aggaaatctc cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac gaccgagcgt
     2581 agcgagtcag tgagcgagga agcggaatat atcctgtatc acatattctg ctgacgcacc
     2641 ggtgcagcct tttttctcct gccacatgaa gcacttcact gacaccctca tcagtgccaa
     2701 catagtaagc cagtatacac tccgctagcg ctgatgtccg gcggtgcttt tgccgttacg
     2761 caccaccccg tcagtagctg aacaggaggg acagagcttt atgcttgtaa accgttttgt
     2821 gaaaaaattt ttaaaataaa aaaggggacc tctagggtcc ccaattaatt agtaatataa
     2881 tctattaaag gtcattcaaa aggtcatcca ccggatcaat tcccctgctc gcgcaggctg
     2941 ggtgccaagc tctcgggtaa catcaaggcc cgatccttgg agcccttgcc ctcccgcacg
     3001 atgatcgtgc cgtgatcgaa aatccagatc cttgacccgc atttgcaaac cctcactgat
     3061 ccgcatgggg ccctcataaa aaatttattt gctttgtgag cggataacaa ttataataga
     3121 ttcaattgtg agcggataac aatttcacac agaattcatt aaagcaacca caattaacta
     3181 tggcttcctc cgaagacgtt atcaaagagt tcatgcgttt caaagttcgt atggaaggtt
     3241 ccgttaacgg tcacgagttc gaaatcgaag gtgaaggtga aggtcgtccg tacgaaggta
     3301 cccagaccgc taaactgaaa gttaccaaag gtggtccgct gccgttcgct tgggacatcc
     3361 tgtccccgca gttccagtac ggttccaaag cttacgttaa acacccggct gacatcccgg
     3421 actacctgaa actgtccttc ccggaaggtt tcaaatggga acgtgttatg aacttcgaag
     3481 acggtggtgt tgttaccgtt acccaggact cctccctgca agacggtgag ttcatctaca
     3541 aagttaaact gcgtggtacc aacttcccgt ccgacggtcc ggttatgcag aaaaaaacca
     3601 tgggttggga agcttccacc gaacgtatgt acccggaaga cggtgctctg aaaggtgaaa
     3661 tcaaaatgcg tctgaaactg aaagacggtg gtcactacga cgctgaagtt aaaaccacct
     3721 acatggctaa aaaaccggtt cagctgccgg gtgcttacaa aaccgacatc aaactggaca
     3781 tcacctccca caacgaagac tacaccatcg ttgaacagta cgaacgtgct gaaggtcgtc
     3841 actccaccgg tgcttaagga tccgcatgcg agctcggtac cccgggtcga cctgcagcca
     3901 agcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct cagaactcca
     3961 tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatccaag
     4021 ctagcttggc gagattttca ggagctaagg aagctaaaga tccgacatat gccgttccat
     4081 acagaagctg ggcgaacaaa cgatgctcgc cttccagaaa accgaggatg cgaaccactt
     4141 catccggggt cagcaccacc ggcaagcgcc gcgacggccg aggtcttccg atctcctgaa
     4201 gccagggcag atccgtgcac agcaccttgc cgtagaagaa cagcaaggcc gccaatgcct
     4261 gacgatgcgt ggagaccgaa accttgcgct cgttcgccag ccaggacaga aatgcctcga
     4321 cttcgctgct gcccaaggtt gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa
     4381 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac
     4441 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
     4501 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
     4561 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg
     4621 gcagtcgccc taaaacaaag ttaaacatca tgagggaagc ggtgatcgcc gaagtatcga
     4681 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg
     4741 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc
     4801 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
     4861 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg
     4921 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag
     4981 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc
     5041 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg
     5101 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
     5161 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt
     5221 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact
     5281 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt
     5341 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc
     5401 actacgtgaa aggcgagatc accaaggtag tcggcaaata atgtctaaca attcgttcaa
     5461 gccgacgccg cttcgcggcg cggcttaact caagcgttag atgcactaag cacataattg
     5521 ctcacagcca aactatcagg tcaagtctgc ttttattatt tttaagcgtg cataataagc
     5581 cctacacaaa ttgggagata tatcatgaaa ggctggcttt ttcttgttat cgcaatagtt
     5641 ggcgaagtaa tcgcaacat
//
