LOCUS       sequence-306827-        5705 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 5705)
  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 5705)
  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..5705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(294..299,300..316,317..322))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      434..979
                     /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     880..899
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1096..1113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2161..2952
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLAPRADQLEEFVHYVKGEITKVVGK"
     promoter        join(3197..3211,3212..3217,3218..3234,3235..3241)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3217..3233
                     /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    3249..3265
                     /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             3303..3980
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      4044..4138
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     protein_bind    4272..4295
                     /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             5023..5046
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(5119..5136)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5289..5375
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5467..5494
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 acatagaata gtgcttatca ctattgcata tagcatctta tctgacacaa ggaaataata
       61 cccttcgctg ttttttgtta taaggtatat atatataagt gtgcagtaca ggccaaataa
      121 aatatttttt atgtagtatc ttaaatcccg caagaggccc ggcagtaccg gcataaccaa
      181 gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga
      241 tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag
      301 cttatcgatg ataagctgtc aaacatgaga attacgactt atatcgtatg gggctgactt
      361 caggtgctac atttgaagag ataaattgca ctgaaatcta gaaatatttt atctgattaa
      421 taagatgatc ttcttgagat cgttttggtc tgcgcgtaat ctcttgctct gaaaacgaaa
      481 aaaccgcctt gcagggcggt ttttcgaagg ttctctgagc taccaactct ttgaaccgag
      541 gtaactggct tggaggagcg cagtcaccaa aacttgtcct ttcagtttag ccttaaccgg
      601 cgcatgactt caagactaac tcctctaaat caattaccag tggctgctgc cagtggtgct
      661 tttgcatgtc tttccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
      721 gactgaacgg ggggttcgtg catacagtcc agcttggagc gaactgccta cccggaactg
      781 agtgtcaggc gtggaatgag acaaacgcgg ccataacagc ggaatgacac cggtaaaccg
      841 aaaggcagga acaggagagc gcacgaggga gccgccaggg ggaaacgcct ggtatcttta
      901 tagtcctgtc gggtttcgcc accactgatt tgagcgtcag atttcgtgat gcttgtcagg
      961 ggggcggagc ctatggaaaa acggctttgc cgcggccctc tcacttccct gttaagtatc
     1021 ttcctggcat cttccaggaa atctccgccc cgttcgtaag ccatttccgc tcgccgcagt
     1081 cgaacgaccg agcgtagcga gtcagtgagc gaggaagcgg aatatatcct gtatcacata
     1141 ttctgctgac gcaccggtgc agcctttttt ctcctgccac atgaagcact tcactgacac
     1201 cctcatcagt gccaacatag taagccagta tacactccgc tagcgctgat gtccggcggt
     1261 gcttttgccg ttacgcacca ccccgtcagt agctgaacag gagggacaga gctttatgct
     1321 tgtaaaccgt tttgtgaaaa aatttttaaa ataaaaaagg ggacctctag ggtccccaat
     1381 taattagtaa tataatctat taaaggtcat tcaaaaggtc atccaccgga tcaattcccc
     1441 tgctcgcgca ggctgggtgc caagctctcg ggtaacatca aggcccgatc cttggagccc
     1501 ttgccctccc gcacgatgat cgtgccgtga tcgaaatcca gatccttgac ccgcagttgc
     1561 aaaccctcac tgatccgcat gcccgttcca tacagaagct gggcgaacaa acgatgctcg
     1621 ccttccagaa aaccgaggat gcgaaccact tcatccgggg tcagcaccac cggcaagcgc
     1681 cgcgacggcc gaggtcttcc gatctcctga agccagggca gatccgtgca cagcaccttg
     1741 ccgtagaaga acagcaaggc cgccaatgcc tgacgatgcg tggagaccga aaccttgcgc
     1801 tcgttcgcca gccaggacag aaatgcctcg acttcgctgc tgcccaaggt tgccgggtga
     1861 cgcacaccgt ggaaacggat gaaggcacga acccagtgga cataagcctg ttcggttcgt
     1921 aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc
     1981 agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta
     2041 cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg
     2101 ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatc
     2161 atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc
     2221 gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc
     2281 ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa
     2341 acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc
     2401 gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt
     2461 tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt
     2521 atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa
     2581 catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag
     2641 gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct
     2701 ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc
     2761 aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat
     2821 cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc
     2881 ccgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta
     2941 gtcggcaaat aatgtctaac aattcgttca agccgacgcc gcttcgcggc gcggcttaac
     3001 tcaagcgtta gatgcactaa gcacataatt gctcacagcc aaactatcag gtcaagtctg
     3061 cttttattat ttttaagcgt gcataataag ccctacacaa attgggagat atatcatgaa
     3121 aggctggctt tttcttgtta tcgcaatagt tggcgaagta atcgcaacat ccgcattaaa
     3181 atctagcgag gggccctcat aaaaaattta tttgctttgt gagcggataa caattataat
     3241 agattcaatt gtgagcggat aacaatttca cacagaattc attaaagcaa ccacaattaa
     3301 ctatggcttc ctccgaagac gttatcaaag agttcatgcg tttcaaagtt cgtatggaag
     3361 gttccgttaa cggtcacgag ttcgaaatcg aaggtgaagg tgaaggtcgt ccgtacgaag
     3421 gtacccagac cgctaaactg aaagttacca aaggtggtcc gctgccgttc gcttgggaca
     3481 tcctgtcccc gcagttccag tacggttcca aagcttacgt taaacacccg gctgacatcc
     3541 cggactacct gaaactgtcc ttcccggaag gtttcaaatg ggaacgtgtt atgaacttcg
     3601 aagacggtgg tgttgttacc gttacccagg actcctccct gcaagacggt gagttcatct
     3661 acaaagttaa actgcgtggt accaacttcc cgtccgacgg tccggttatg cagaaaaaaa
     3721 ccatgggttg ggaagcttcc accgaacgta tgtacccgga agacggtgct ctgaaaggtg
     3781 aaatcaaaat gcgtctgaaa ctgaaagacg gtggtcacta cgacgctgaa gttaaaacca
     3841 cctacatggc taaaaaaccg gttcagctgc cgggtgctta caaaaccgac atcaaactgg
     3901 acatcacctc ccacaacgaa gactacacca tcgttgaaca gtacgaacgt gctgaaggtc
     3961 gtcactccac cggtgcttaa ggatccgcat gcgagctcgg taccccgggt cgacctgcag
     4021 ccaagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga cctcagaact
     4081 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     4141 aagctagctt ggcgagattt tcaggagcta aggaagctaa agatccgagg cgcgcccaga
     4201 gattttgaga cacaacgtgg ctttccatca aaaaaatatt gacaacataa aaaactttgt
     4261 gttatacttg tggaattgtg agcggataac aattctatat ctgttatttt ttccaaccac
     4321 agatctatga gcaaaggtga agaactgttt accggcgttg tgccgattct ggtggaactg
     4381 gatggcgatg tgaacggtca caaattcagc gtgcgtggtg aaggtgaagg cgatgccacg
     4441 attggcaaac tgacgctgaa atttatctgc accaccggca aactgccggt gccgtggccg
     4501 acgctggtga ccaccctgac ctatggcgtt cagtgtttta gtcgctatcc ggatcacatg
     4561 aaacgtcacg atttctttaa atctgcaatg ccggaaggct atgtgcagga acgtacgatt
     4621 agctttaaag atgatggcaa atataaaacg cgcgccgttg tgaaatttga aggcgatacc
     4681 ctggtgaacc gcattgaact gaaaggcacg gattttaaag aagatggcaa tatcctgggc
     4741 cataaactgg aatacaactt taatagccat aatgtttata ttacggcgga taaacagaaa
     4801 aatggcatca aagcgaattt taccgttcgc cataacgttg aagatggcag tgtgcagctg
     4861 gcagatcatt atcagcagaa taccccgatt ggtgatggtc cggtgctgct gccggataat
     4921 cattatctga gcacgcagac cgttctgtct aaagatccga acgaaaaagg cacgcgggac
     4981 cacatggttc tgcacgaata tgtgaatgcg gcaggtatta tgtggagcca tccgcagttc
     5041 gaaaaataag tcgaccggct gctaacaaag cccgcggccg ctgaagatcg atctcgacga
     5101 gtgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     5161 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     5221 agtgaaacgc cgtagcgccg atggtagtgt ggggtcaccc catgcgagag tagggaactg
     5281 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     5341 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     5401 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     5461 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacactcc taggggacgc
     5521 cactctagga aatccgccat aaaacaaaag gctcagtcgg aagactgggc cttttgtttt
     5581 atgtcgacgg gcataaatag gtttaatttt gctacggggg cgttatttag gttttttctt
     5641 ctttcgaaaa aatctttctt tatgaagtta aaagctatgt attcaatagc atattttgaa
     5701 tatgg
//
