LOCUS       sequence-332750-        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             28..36
                     /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             52..69
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(1176..1209)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1261..1971)
                     /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(1984..2001)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2017..2025
                     /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(2754..2771)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2787..2795
                     /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(2818..3513)
                     /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     3362..3380
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(3529..3546)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     rep_origin      4776..5364
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5265..5284
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(6622..6655)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     6782..6801
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6782..6800
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             7588..7605
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(7627..8318,1..16)
                     /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 cgagctgtac aagtaagctt gcttaattaa ggaggtatct ccatgagcgg tcaccatcac
       61 catcaccacg gcggtagcgc catgagcagc gtgagcaagg gcgaggagac cacaatgggc
      121 gtgatcaagc cggacatgaa gatcaagctg aagatggagg gcaacgtgaa cggccacgcc
      181 ttcgtgatcg agggcgaggg cgagggcaag ccgtacgacg gcaccaacac catcaacctg
      241 gaggtgaagg agggcgcccc gctgccgttc tcgtacgaca tcctgaccac cgccttctcg
      301 tacggcaacc gcgccttcac caagtacccg gacgacatcc cgaactactt caagcagtcg
      361 ttcccggagg gctactcgtg ggagcgcacc atgaccttcg aggacaaggg catcgtgaag
      421 gtgaagtcgg acatctcgat ggaggaggac tcgttcatct acgagatcca cctgaagggc
      481 gagaacttcc cgccgaacgg cccggtgatg cagaaggaga ccaccggctg ggacgcctcg
      541 accgagcgca tgtacgtgcg cgacggcgtg ctgaagggcg acgtgaagat gaagctgctg
      601 ctggagggcg gcggccacca ccgcgtggac ttcaagacca tctaccgcgc caagaaggcc
      661 gtgaagctgc cggactacca cttcgtggac caccgcatcg agatcctgaa ccacgacaag
      721 gactacaaca aggtgaccgt gtacgagatc gccgtggccc gcaactcgac cgacggcatg
      781 gacgagctgt acaagtaatc gataaaaaaa aaagccccgc aatcgcgggg cttttttttt
      841 tacgcgtcgc cgacctacac gtattcgcag agttaagcgg tcacccccgc cggtttgacc
      901 ccaattttga ccccaattgc atcggggttc ggggcgattc gccgatgtcc gccattgtgc
      961 gcaaccgggc gtgacctgcg gattcggtac ccgggcggat ccacgcggat gccttttgaa
     1021 tgactggggg tcaaggggtc gcaggttcaa atcctgtcag cccgacgcag atcaggggcg
     1081 gttttccgga gacgggaagc cgcccctttt tctgtttgac cccaattttg accccaattg
     1141 ctccggcgtc cgtccgcgtc cgtcgagggc gcgccataac ttcgtatagc atacattata
     1201 cgaagttatg atatcaaaaa aaagcccgcc attaggcggg ctcaattgag cagatcttta
     1261 cttgtacagc tcgtccatgc caccaccaag gttgctgtat ttggcaactg ccacttctct
     1321 ttgcactacg taggtttcat tgtcactctc ctcgatcctt tccaggcggt gatcaacggc
     1381 atggacaccg ggcatcttga tgttcccgac ggtctttttt gacctgtaag ttgtcacgaa
     1441 gttgcagtgc agatggccac caccatcaac tttcagtgcg atgtcagtgt atcctctcag
     1501 accaccatct gctggataca tcatctctgt attaggctcc caacccttgg tcttcttctg
     1561 catcacggga ccattggagg gaaagtttac ccctctgacc ttgacgttgt agacgagctc
     1621 gccatcctca aggctggtgt cctgcgtgac ggtgacgact ccaccatctt cgtatctcgt
     1681 aactctttcc caagtaaaac cctcaggaaa ggactgttta aagaaatcag ggatgtcggc
     1741 cgggtacttg ataaaggtac ggctgccata catgaacgac gtggcaagaa tgtcaaaggc
     1801 aaatggcagg ggtcctccct cgatgacttt gatcctcatg gtttgcactc cctcgtacgg
     1861 tctgccttct ccttcacctg tgcatttgaa ttggtggccg ttgaccgaac cttccatgac
     1921 caccttcata cgcatatttt ccttgatcag ctcttcgccc ttagacacca tggcgctacc
     1981 gccgtggtga tggtgatggt gaccgctcat ggagatacct ccttaattaa aacgataagc
     2041 tttcacttat acaattcatc catccccccg gtggaatgac gaccctccga gcgttcgtat
     2101 tgttccacca ccgtgtaatc ttcgttatgg ctcgtaatat ccagcttacg gtcgacatta
     2161 tacgccccgg gcatttgcac gggttttttg gctttgtagg tggttttgaa gtccgccaag
     2221 tagcgcccac catctttcaa ccgcagggcc attttaatgt cacccttcaa cacgccatct
     2281 tctggataca gccgctcggt cgaggcctcc cagcccatcg ttttcttttg catgacggga
     2341 ccgtcggggg ggaaattcgt cccccgcaac ttgaccttat agatgagggt cccgtcttcc
     2401 agcgaggtat cttgggtgac cgtcaccgca cccccgtcct caaaattcat cacacgttcc
     2461 catttgaacc cctcggggaa gctctgtttg tagtagtccg ggatgtcggc ggggtgcttg
     2521 gtaaacgcac gcgaaccgta catgaattgt gggctgagga tatcccacga gaacgggagc
     2581 gggccgcctt tcgtgacctt caatttcgcg gtttgcgtcc cttcgtatgg acggccctcg
     2641 ccctccccct cgatttcaaa ctcatgcccg ttcatcgacc cttccatatg cactttgaaa
     2701 cgcataaact ctttaatcac cgcctcgccc ttgctcacca tggcgctacc gccgtggtga
     2761 tggtgatggt gaccgctcat ggagatacct ccttaattag catgcggaaa gctttcactt
     2821 gtacagctcg tccatgccgc cggtggagtg gcggccctcg gagcgttcgt actgttccac
     2881 cacggtgtag tcctcgttgt gggaggtgat gtccaacttg cggtcgacgt tgtaggcgcc
     2941 gggcatctgc acgggcttct tggccttgta ggtggtcttg aagtccgcca ggtagcggcc
     3001 gccgtccttc aggcgcaggg ccatcttaat gtcgcccttc agcacgccgt cctcggggta
     3061 caaccgctcg gtggacgctt cccagcccat tgtcttcttc tgcattacgg ggccgtcagg
     3121 agggaagttg gtgccgcgga gcttcacctt gtagatcagg gtgccgtcct ccagggaggt
     3181 gtcctgggtc acggtcacgg cgccgccgtc ctcgaagttc atcacgcgct cccacttgaa
     3241 gccctcgggg aaggactgct tatagtagtc ggggatgtcg gcggggtgct tggtgaaggc
     3301 cctggagccg tacatgaact gaggggacag gatgtcccag gagaagggca gggggccacc
     3361 cttggtcacc ttcagcttgg cggtctgggt gccctcgtag gggcggccct cgccctcgcc
     3421 ctcgatctcg aactcgtggc cgttcatgga gccctccatg tgcaccttga accgcatgaa
     3481 ctccttgatc actgcctcgc ccttgctcac catgggcccg ctaccgccgt ggtgatggtg
     3541 atggtgaccg ctcatctctg tcccccttcc gagccagcca actcggctgc atgtgtcggg
     3601 ccgccggctt tgtcacctca ggctgcctcg cagagctgcg agccaaggtg gatgtcaacc
     3661 gtgaagttgc tgtgaaaagc cggacccctc gggcgcaatg cggttaccca ggcaaatcgg
     3721 gcagtggctt ggagctgccg gccggcgctg ccagccggta cgacgctgtg acccgggtgc
     3781 gctggtagag ccaagcgagg aggccggcaa agacgatggc tcccagcggc tgggaggcaa
     3841 acctgaatcc gtcgccgacc atggccagcg gctcctcttt gccagttgtc gcgacgatga
     3901 cggcaaagag caccccgtat aggctttcgc ccacaatcag gcccgtcgcg agcatgacgc
     3961 ccaaccgttt cttgcgctcg tcgtcatccc cagaccaccg cgcccaggag tcatagatcc
     4021 gcccgaggaa tgcgccgatc gggatcatca gcgtcagtgc ggccgacagg tacataccca
     4081 tacccaccgc tagtggcggc agccgaaggt tggttgtcgt cttggccagt gtctcgtcga
     4141 gcgcgaccgc tatcacgccg gtcaaggccc ctacaccgac cagcgaccag ttcagcgagc
     4201 caccaaatac tcccttggcc agcgcggaca tgagcgcggc ttgcggggcg gccaatgcgt
     4261 tggccgttgc gcccggcgcc ccctggaacc cgaatccagc ctgcatcagc tgcaggatcg
     4321 gcgccatcac caccgacccg acgagcacgc cgatgatcag tgcaacctgc tgcttccatg
     4381 gggtagcgcc gacgagttgg ccggttttga ggtcctgcag attgtcgttg gaaatagtcg
     4441 ccacgccgaa gaccaatgca gcggtaaaca cggtgtaggc caccagggcc ggaatctgcg
     4501 agccggtggc cggaccatac gcagtcttga tcagcagacc ggccgctagc cgagaatggg
     4561 agaccctgca ccacggcaat ccaataattg tagaaatatt ggtaactgtc agaccaagtt
     4621 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4681 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4741 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4801 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4861 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4921 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4981 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5041 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5101 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5161 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5221 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5281 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5341 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5401 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5461 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     5521 gagtcagtga gcgaggaagc cgacggtatc gggtaccctc gagccggccg tgcggaatta
     5581 agccggcccg taccctgtga atagaggtcc gctgtgacac aagaatccct gttacttctc
     5641 gaccgtattg attcggatga ttcctacgcg agcctgcgga acgaccagga attctgggag
     5701 ccgctggccc gccgagccct ggaggagctc gggctgccgg tgccgccggt gctgcgggtg
     5761 cccggcgaga gcaccaaccc cgtactggtc ggcgagcccg gcccggtgat caagctgttc
     5821 ggcgagcact ggtgcggtcc ggagagcctc gcgtcggagt cggaggcgta cgcggtcctg
     5881 gcggacgccc cggtgccggt gccccgcctc ctcggccgcg gcgagctgcg gcccggcacc
     5941 ggagcctggc cgtggcccta cctggtgatg agccggatga ccggcaccac ctggcggtcc
     6001 gcgatggacg gcacgaccga ccggaacgcg ctgctcgccc tggcccgcga actcggccgg
     6061 gtgctcggcc ggctgcacag ggtgccgctg accgggaaca ccgtgctcac cccccattcc
     6121 gaggtcttcc cggaactgct gcgggaacgc cgcgcggcga ccgtcgagga ccaccgcggg
     6181 tggggctacc tctcgccccg gctgctggac cgcctggagg actggctgcc ggacgtggac
     6241 acgctgctgg ccggccgcga accccggttc gtccacggcg acctgcacgg gaccaacatc
     6301 ttcgtggacc tggccgcgac cgaggtcacc gggatcgtcg acttcaccga cgtctatgcg
     6361 ggagactccc gctacagcct ggtgcaactg catctcaacg ccttccgggg cgaccgcgag
     6421 atcctggccg cgctgctcga cggggcgcag tggaagcgga ccgaggactt cgcccgcgaa
     6481 ctgctcgcct tcaccttcct gcacgacttc gaggtgttcg aggagacccc gctggatctc
     6541 tccggcttca ccgatccgga ggaactggcg cagttcctct gggggccgcc ggacaccgcc
     6601 cccggcgcct gacgccccgg gataacttcg tatagcatac attatacgaa gttatatgca
     6661 ttgactaagc tagatacctg aaaaaaaaaa agcgccgcag ttgcggcgct tttttttttc
     6721 tcgactagtg tttaaactct agacgatgat aagcggtcaa acatgagaat tcgcggccgc
     6781 ataatacgac tcactatagg gatcttaatt aaggcgcctc atgttctttc ctgcgttatc
     6841 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     6901 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     6961 accgcctctc ccagatctga tatcgctaga ggaaacagct atgaccatga ttacgccaag
     7021 cttgcatgcc tgcagctagg gcaccaattt gcgattaggg cttgacagcc acccggccag
     7081 tagtgcattc ttgtgtcacc gcaagcacct cgctaggtga ggcgtctgcg cggatatagg
     7141 ccactgacct cgaacgtcga aagacgccca gggtcaggac agctcttccc ggcttaaggg
     7201 ttgagcccaa gtggcttccg gctggaccgg ccggatacgc cgtgtggtgc caaagctctg
     7261 acgagagggg tgccgagttc ggtggtctgc tgggctgtca tccctttgtg ctgtgcatcg
     7321 gcatccccgt gtgccccggc cgtgaggagg tgagagcgaa atgagtcccg gcgatagtcc
     7381 gtatccgaga tcgacgaccg tttcgttccg atccgacccc ggcgccgttt tcgcactctg
     7441 aatcggcctt ccggttcgaa atccgttatt tcgcaagctc gttgcttcgc ggccttgtgt
     7501 gagtgacgtt cacgggaagt agccacgaca gaagcggtca taggcctccg ggttcggtcg
     7561 tctgtcagga gaagacccat gagcggtcac catcaccatc accacggcgg tagcgccatg
     7621 ggcagcgtga gcaagggcga ggagaccaca atgggcgtaa tcaagcccga catgaagatc
     7681 aagctgaaga tggagggcaa cgtgaatggc cacgccttcg tgatcgaggg cgagggcgag
     7741 ggcaagccct acgacggcac caacaccatc aacctggagg tgaaggaggg agcccccctg
     7801 cccttctcct acgacattct gaccaccgcg ttcagttacg gcaacagggc cttcaccaag
     7861 taccccgacg acatccccaa ctacttcaag cagtccttcc ccgagggcta ctcttgggag
     7921 cgcaccatga ccttcgagga caagggcatc gtgaaggtga agtccgacat ctcgatggag
     7981 gaggactcct tcatctacga gatacacctc aagggcgaga acttcccccc caacggcccc
     8041 gtgatgcaga aggagaccac cggctgggac gcctccaccg agaggatgta cgtgcgcgac
     8101 ggcgtgctga agggcgacgt caagatgaag ctgctgctgg agggcggcgg ccaccaccgc
     8161 gttgacttca agaccatcta cagggccaag aaggcggtga agctgcccga ctatcacttt
     8221 gtggaccacc gcatcgagat cctgaaccac gacaaggact acaacaaggt gaccgtttac
     8281 gagatcgccg tggcccgcaa ctccaccgac ggcatgga
//
