LOCUS       sequence-329427-        8318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tri-Responsive Reporter Gene Construct for redgreen, triple red, and
            double green.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8318)
  AUTHORS   Kolbe K, Bell AC, Prosser GA, Assmann M, Yang HJ, Forbes HE, 
            Gallucci S, Mayer-Barber KD, Boshoff HI, Barry Iii CE
  TITLE     Development and Optimization of Chromosomally-Integrated Fluorescent
            Mycobacterium tuberculosis Reporter Constructs.
  JOURNAL   Front Microbiol. 2020 Dec 9;11:591866. doi: 
            10.3389/fmicb.2020.591866. eCollection 2020.
  PUBMED    33362741
REFERENCE   2  (bases 1 to 8318)
  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: "Front 
            Microbiol."; date: "2020-12-9"; volume: "11:591866. doi"; pages: " 
            10.3389/fmicb.2020.591866. eCollection 2020"
COMMENT     Sequence Label: pGilesSHdI_redgreen_2395A_His6_triple
            red_pLef...
FEATURES             Location/Qualifiers
     source          1..8318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..20
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             36..53
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(1160..1193)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1245..1955)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     CDS             complement(1968..1985)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2001..2009
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             complement(2738..2755)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2771..2779
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             complement(2802..3497)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     3346..3364
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(3513..3530)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     rep_origin      4760..5348
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5249..5268
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(6606..6639)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     6766..6785
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6766..6784
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             7572..7589
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7611..8318
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="VSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDGT
                     NTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTFE
                     DKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLKG
                     DVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIAV
                     ARNSTDGMDELYK"
ORIGIN
        1 gcttgcttaa ttaaggaggt atctccatga gcggtcacca tcaccatcac cacggcggta
       61 gcgccatgag cagcgtgagc aagggcgagg agaccacaat gggcgtgatc aagccggaca
      121 tgaagatcaa gctgaagatg gagggcaacg tgaacggcca cgccttcgtg atcgagggcg
      181 agggcgaggg caagccgtac gacggcacca acaccatcaa cctggaggtg aaggagggcg
      241 ccccgctgcc gttctcgtac gacatcctga ccaccgcctt ctcgtacggc aaccgcgcct
      301 tcaccaagta cccggacgac atcccgaact acttcaagca gtcgttcccg gagggctact
      361 cgtgggagcg caccatgacc ttcgaggaca agggcatcgt gaaggtgaag tcggacatct
      421 cgatggagga ggactcgttc atctacgaga tccacctgaa gggcgagaac ttcccgccga
      481 acggcccggt gatgcagaag gagaccaccg gctgggacgc ctcgaccgag cgcatgtacg
      541 tgcgcgacgg cgtgctgaag ggcgacgtga agatgaagct gctgctggag ggcggcggcc
      601 accaccgcgt ggacttcaag accatctacc gcgccaagaa ggccgtgaag ctgccggact
      661 accacttcgt ggaccaccgc atcgagatcc tgaaccacga caaggactac aacaaggtga
      721 ccgtgtacga gatcgccgtg gcccgcaact cgaccgacgg catggacgag ctgtacaagt
      781 aatcgataaa aaaaaaagcc ccgcaatcgc ggggcttttt tttttacgcg tcgccgacct
      841 acacgtattc gcagagttaa gcggtcaccc ccgccggttt gaccccaatt ttgaccccaa
      901 ttgcatcggg gttcggggcg attcgccgat gtccgccatt gtgcgcaacc gggcgtgacc
      961 tgcggattcg gtacccgggc ggatccacgc ggatgccttt tgaatgactg ggggtcaagg
     1021 ggtcgcaggt tcaaatcctg tcagcccgac gcagatcagg ggcggttttc cggagacggg
     1081 aagccgcccc tttttctgtt tgaccccaat tttgacccca attgctccgg cgtccgtccg
     1141 cgtccgtcga gggcgcgcca taacttcgta tagcatacat tatacgaagt tatgatatca
     1201 aaaaaaagcc cgccattagg cgggctcaat tgagcagatc tttacttgta cagctcgtcc
     1261 atgccaccac caaggttgct gtatttggca actgccactt ctctttgcac tacgtaggtt
     1321 tcattgtcac tctcctcgat cctttccagg cggtgatcaa cggcatggac accgggcatc
     1381 ttgatgttcc cgacggtctt ttttgacctg taagttgtca cgaagttgca gtgcagatgg
     1441 ccaccaccat caactttcag tgcgatgtca gtgtatcctc tcagaccacc atctgctgga
     1501 tacatcatct ctgtattagg ctcccaaccc ttggtcttct tctgcatcac gggaccattg
     1561 gagggaaagt ttacccctct gaccttgacg ttgtagacga gctcgccatc ctcaaggctg
     1621 gtgtcctgcg tgacggtgac gactccacca tcttcgtatc tcgtaactct ttcccaagta
     1681 aaaccctcag gaaaggactg tttaaagaaa tcagggatgt cggccgggta cttgataaag
     1741 gtacggctgc catacatgaa cgacgtggca agaatgtcaa aggcaaatgg caggggtcct
     1801 ccctcgatga ctttgatcct catggtttgc actccctcgt acggtctgcc ttctccttca
     1861 cctgtgcatt tgaattggtg gccgttgacc gaaccttcca tgaccacctt catacgcata
     1921 ttttccttga tcagctcttc gcccttagac accatggcgc taccgccgtg gtgatggtga
     1981 tggtgaccgc tcatggagat acctccttaa ttaaaacgat aagctttcac ttatacaatt
     2041 catccatccc cccggtggaa tgacgaccct ccgagcgttc gtattgttcc accaccgtgt
     2101 aatcttcgtt atggctcgta atatccagct tacggtcgac attatacgcc ccgggcattt
     2161 gcacgggttt tttggctttg taggtggttt tgaagtccgc caagtagcgc ccaccatctt
     2221 tcaaccgcag ggccatttta atgtcaccct tcaacacgcc atcttctgga tacagccgct
     2281 cggtcgaggc ctcccagccc atcgttttct tttgcatgac gggaccgtcg ggggggaaat
     2341 tcgtcccccg caacttgacc ttatagatga gggtcccgtc ttccagcgag gtatcttggg
     2401 tgaccgtcac cgcacccccg tcctcaaaat tcatcacacg ttcccatttg aacccctcgg
     2461 ggaagctctg tttgtagtag tccgggatgt cggcggggtg cttggtaaac gcacgcgaac
     2521 cgtacatgaa ttgtgggctg aggatatccc acgagaacgg gagcgggccg cctttcgtga
     2581 ccttcaattt cgcggtttgc gtcccttcgt atggacggcc ctcgccctcc ccctcgattt
     2641 caaactcatg cccgttcatc gacccttcca tatgcacttt gaaacgcata aactctttaa
     2701 tcaccgcctc gcccttgctc accatggcgc taccgccgtg gtgatggtga tggtgaccgc
     2761 tcatggagat acctccttaa ttagcatgcg gaaagctttc acttgtacag ctcgtccatg
     2821 ccgccggtgg agtggcggcc ctcggagcgt tcgtactgtt ccaccacggt gtagtcctcg
     2881 ttgtgggagg tgatgtccaa cttgcggtcg acgttgtagg cgccgggcat ctgcacgggc
     2941 ttcttggcct tgtaggtggt cttgaagtcc gccaggtagc ggccgccgtc cttcaggcgc
     3001 agggccatct taatgtcgcc cttcagcacg ccgtcctcgg ggtacaaccg ctcggtggac
     3061 gcttcccagc ccattgtctt cttctgcatt acggggccgt caggagggaa gttggtgccg
     3121 cggagcttca ccttgtagat cagggtgccg tcctccaggg aggtgtcctg ggtcacggtc
     3181 acggcgccgc cgtcctcgaa gttcatcacg cgctcccact tgaagccctc ggggaaggac
     3241 tgcttatagt agtcggggat gtcggcgggg tgcttggtga aggccctgga gccgtacatg
     3301 aactgagggg acaggatgtc ccaggagaag ggcagggggc cacccttggt caccttcagc
     3361 ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg
     3421 tggccgttca tggagccctc catgtgcacc ttgaaccgca tgaactcctt gatcactgcc
     3481 tcgcccttgc tcaccatggg cccgctaccg ccgtggtgat ggtgatggtg accgctcatc
     3541 tctgtccccc ttccgagcca gccaactcgg ctgcatgtgt cgggccgccg gctttgtcac
     3601 ctcaggctgc ctcgcagagc tgcgagccaa ggtggatgtc aaccgtgaag ttgctgtgaa
     3661 aagccggacc cctcgggcgc aatgcggtta cccaggcaaa tcgggcagtg gcttggagct
     3721 gccggccggc gctgccagcc ggtacgacgc tgtgacccgg gtgcgctggt agagccaagc
     3781 gaggaggccg gcaaagacga tggctcccag cggctgggag gcaaacctga atccgtcgcc
     3841 gaccatggcc agcggctcct ctttgccagt tgtcgcgacg atgacggcaa agagcacccc
     3901 gtataggctt tcgcccacaa tcaggcccgt cgcgagcatg acgcccaacc gtttcttgcg
     3961 ctcgtcgtca tccccagacc accgcgccca ggagtcatag atccgcccga ggaatgcgcc
     4021 gatcgggatc atcagcgtca gtgcggccga caggtacata cccataccca ccgctagtgg
     4081 cggcagccga aggttggttg tcgtcttggc cagtgtctcg tcgagcgcga ccgctatcac
     4141 gccggtcaag gcccctacac cgaccagcga ccagttcagc gagccaccaa atactccctt
     4201 ggccagcgcg gacatgagcg cggcttgcgg ggcggccaat gcgttggccg ttgcgcccgg
     4261 cgccccctgg aacccgaatc cagcctgcat cagctgcagg atcggcgcca tcaccaccga
     4321 cccgacgagc acgccgatga tcagtgcaac ctgctgcttc catggggtag cgccgacgag
     4381 ttggccggtt ttgaggtcct gcagattgtc gttggaaata gtcgccacgc cgaagaccaa
     4441 tgcagcggta aacacggtgt aggccaccag ggccggaatc tgcgagccgg tggccggacc
     4501 atacgcagtc ttgatcagca gaccggccgc tagccgagaa tgggagaccc tgcaccacgg
     4561 caatccaata attgtagaaa tattggtaac tgtcagacca agtttactca tatatacttt
     4621 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4681 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4741 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4801 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4861 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4921 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4981 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5041 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5101 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5161 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5221 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5281 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5341 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5401 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5461 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5521 aagccgacgg tatcgggtac cctcgagccg gccgtgcgga attaagccgg cccgtaccct
     5581 gtgaatagag gtccgctgtg acacaagaat ccctgttact tctcgaccgt attgattcgg
     5641 atgattccta cgcgagcctg cggaacgacc aggaattctg ggagccgctg gcccgccgag
     5701 ccctggagga gctcgggctg ccggtgccgc cggtgctgcg ggtgcccggc gagagcacca
     5761 accccgtact ggtcggcgag cccggcccgg tgatcaagct gttcggcgag cactggtgcg
     5821 gtccggagag cctcgcgtcg gagtcggagg cgtacgcggt cctggcggac gccccggtgc
     5881 cggtgccccg cctcctcggc cgcggcgagc tgcggcccgg caccggagcc tggccgtggc
     5941 cctacctggt gatgagccgg atgaccggca ccacctggcg gtccgcgatg gacggcacga
     6001 ccgaccggaa cgcgctgctc gccctggccc gcgaactcgg ccgggtgctc ggccggctgc
     6061 acagggtgcc gctgaccggg aacaccgtgc tcacccccca ttccgaggtc ttcccggaac
     6121 tgctgcggga acgccgcgcg gcgaccgtcg aggaccaccg cgggtggggc tacctctcgc
     6181 cccggctgct ggaccgcctg gaggactggc tgccggacgt ggacacgctg ctggccggcc
     6241 gcgaaccccg gttcgtccac ggcgacctgc acgggaccaa catcttcgtg gacctggccg
     6301 cgaccgaggt caccgggatc gtcgacttca ccgacgtcta tgcgggagac tcccgctaca
     6361 gcctggtgca actgcatctc aacgccttcc ggggcgaccg cgagatcctg gccgcgctgc
     6421 tcgacggggc gcagtggaag cggaccgagg acttcgcccg cgaactgctc gccttcacct
     6481 tcctgcacga cttcgaggtg ttcgaggaga ccccgctgga tctctccggc ttcaccgatc
     6541 cggaggaact ggcgcagttc ctctgggggc cgccggacac cgcccccggc gcctgacgcc
     6601 ccgggataac ttcgtatagc atacattata cgaagttata tgcattgact aagctagata
     6661 cctgaaaaaa aaaaagcgcc gcagttgcgg cgcttttttt tttctcgact agtgtttaaa
     6721 ctctagacga tgataagcgg tcaaacatga gaattcgcgg ccgcataata cgactcacta
     6781 tagggatctt aattaaggcg cctcatgttc tttcctgcgt tatcccctga ttctgtggat
     6841 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6901 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctcccagat
     6961 ctgatatcgc tagaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagc
     7021 tagggcacca atttgcgatt agggcttgac agccacccgg ccagtagtgc attcttgtgt
     7081 caccgcaagc acctcgctag gtgaggcgtc tgcgcggata taggccactg acctcgaacg
     7141 tcgaaagacg cccagggtca ggacagctct tcccggctta agggttgagc ccaagtggct
     7201 tccggctgga ccggccggat acgccgtgtg gtgccaaagc tctgacgaga ggggtgccga
     7261 gttcggtggt ctgctgggct gtcatccctt tgtgctgtgc atcggcatcc ccgtgtgccc
     7321 cggccgtgag gaggtgagag cgaaatgagt cccggcgata gtccgtatcc gagatcgacg
     7381 accgtttcgt tccgatccga ccccggcgcc gttttcgcac tctgaatcgg ccttccggtt
     7441 cgaaatccgt tatttcgcaa gctcgttgct tcgcggcctt gtgtgagtga cgttcacggg
     7501 aagtagccac gacagaagcg gtcataggcc tccgggttcg gtcgtctgtc aggagaagac
     7561 ccatgagcgg tcaccatcac catcaccacg gcggtagcgc catgggcagc gtgagcaagg
     7621 gcgaggagac cacaatgggc gtaatcaagc ccgacatgaa gatcaagctg aagatggagg
     7681 gcaacgtgaa tggccacgcc ttcgtgatcg agggcgaggg cgagggcaag ccctacgacg
     7741 gcaccaacac catcaacctg gaggtgaagg agggagcccc cctgcccttc tcctacgaca
     7801 ttctgaccac cgcgttcagt tacggcaaca gggccttcac caagtacccc gacgacatcc
     7861 ccaactactt caagcagtcc ttccccgagg gctactcttg ggagcgcacc atgaccttcg
     7921 aggacaaggg catcgtgaag gtgaagtccg acatctcgat ggaggaggac tccttcatct
     7981 acgagataca cctcaagggc gagaacttcc cccccaacgg ccccgtgatg cagaaggaga
     8041 ccaccggctg ggacgcctcc accgagagga tgtacgtgcg cgacggcgtg ctgaagggcg
     8101 acgtcaagat gaagctgctg ctggagggcg gcggccacca ccgcgttgac ttcaagacca
     8161 tctacagggc caagaaggcg gtgaagctgc ccgactatca ctttgtggac caccgcatcg
     8221 agatcctgaa ccacgacaag gactacaaca aggtgaccgt ttacgagatc gccgtggccc
     8281 gcaactccac cgacggcatg gacgagctgt acaagtaa
//
