LOCUS       sequence-332735-        5463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis 
            Reporter Constructs for triple red.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5463)
  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 5463)
  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: Plasmid L5 attB:Psmyc triple red
FEATURES             Location/Qualifiers
     source          1..5463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(578..597)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             589..1404
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(662..681)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1735..2323
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2224..2243
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             2823..2831
                     /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             2847..2864
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2877..3572
                     /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     complement(3010..3028)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     RBS             3595..3603
                     /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             3619..3636
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4365..4373
                     /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             4389..4406
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4419..5129
                     /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"
     terminator      5185..5231
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cgtatgccca ggtcagaaga gtcgcacaag agttgcagac ccctggaaag aaaaatggcc
       61 agagggcgaa aacaccctct gaccagcgga gcgggcgacg ggaatcgaac ccgcgtagct
      121 agtttggaag aatgggtgtc tgccgaccac atatatgggc cggtcaagat aggtttttac
      181 cccctctcgg ctgcatcctc taagtggaaa gaaattgcag gtcgtagaag cgcgttgaag
      241 cctgagagtt gcacaggagt tgcaacccgg tagccttgtt cacgacgaga ggagacctag
      301 ggatcctccc aaggacactg agtcctaaag aggggggttt cttgtcagta cgcgaagaac
      361 cacgcctggc cgcgagcgcc agcaccgccg ctctgtgcgg agacctgggc accagccccg
      421 ccgccgccag gagcattgcc gttcccgcca gctagccacc tgacgtcggg ggggggggag
      481 agccacgttg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
      541 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
      601 caacgggaaa cgtcttgctc gaggccgcga ttaaattcca acatggatgc tgatttatat
      661 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
      721 gggaagcccc atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
      781 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
      841 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa
      901 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
      961 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     1021 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     1081 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataat
     1141 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     1201 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     1261 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     1321 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     1381 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     1441 attacgctga cttgacggga cggcggcttt gttgaataaa tcgaactttt gctgagttga
     1501 aggatcagat cacgcatctt cccgacaacg cagaccgttc cgtggcaaag caaaagttca
     1561 aaatcaccaa ctggtccacc tacaacaaag ctctcaccaa ccgtggctcc ctcactttct
     1621 ggctggatga tggggcgatt caggcctggt atgagtcagc aacaccttct tcacgaggca
     1681 gacctcacta gttccatgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     1741 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1801 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1861 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     1921 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1981 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2041 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2101 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aggggagaaa
     2161 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2221 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2281 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2341 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2401 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2461 ccgaacgacc gagcgcaacg cgtgcggccg cggtaccaga tctttaaatc tagaggatcg
     2521 tcggcaccgt cacggccgtg ggaggcggca cgatccgcga cgtgatgatc ggccgcatcc
     2581 ccacggtgct gcgcagtgag ctctacgcca tcccggcgtt gatctgtgcg ttcgcacgca
     2641 caggcccggt gtgagaaggg tctctgacga gcgggagaac ccacccgggg tgggcgagtt
     2701 tgtcctgcgt gtgctcggtc gagtaggctc tgggagtacc cgtgtgtacg accagcacgg
     2761 catacatcat ttcgacgccg agagattcgc cgcccgaaat gagcacgatc cgcatgctta
     2821 attaaggagg tatctccatg agcggtcacc atcaccatca ccacggcggt agcgccatgg
     2881 tgagcaaggg cgaggcagtg atcaaggagt tcatgcggtt caaggtgcac atggagggct
     2941 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     3001 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttctcc tgggacatcc
     3061 tgtcccctca gttcatgtac ggctccaggg ccttcaccaa gcaccccgcc gacatccccg
     3121 actactataa gcagtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     3181 acggcggcgc cgtgaccgtg acccaggaca cctccctgga ggacggcacc ctgatctaca
     3241 aggtgaagct ccgcggcacc aacttccctc ctgacggccc cgtaatgcag aagaagacaa
     3301 tgggctggga agcgtccacc gagcggttgt accccgagga cggcgtgctg aagggcgaca
     3361 ttaagatggc cctgcgcctg aaggacggcg gccgctacct ggcggacttc aagaccacct
     3421 acaaggccaa gaagcccgtg cagatgcccg gcgcctacaa cgtcgaccgc aagttggaca
     3481 tcacctccca caacgaggac tacaccgtgg tggaacagta cgaacgctcc gagggccgcc
     3541 actccaccgg cggcatggac gagctgtaca agtgaaagct ttccgcatgc taattaagga
     3601 ggtatctcca tgagcggtca ccatcaccat caccacggcg gtagcgccat ggtgagcaag
     3661 ggcgaggcgg tgattaaaga gtttatgcgt ttcaaagtgc atatggaagg gtcgatgaac
     3721 gggcatgagt ttgaaatcga gggggagggc gagggccgtc catacgaagg gacgcaaacc
     3781 gcgaaattga aggtcacgaa aggcggcccg ctcccgttct cgtgggatat cctcagccca
     3841 caattcatgt acggttcgcg tgcgtttacc aagcaccccg ccgacatccc ggactactac
     3901 aaacagagct tccccgaggg gttcaaatgg gaacgtgtga tgaattttga ggacgggggt
     3961 gcggtgacgg tcacccaaga tacctcgctg gaagacggga ccctcatcta taaggtcaag
     4021 ttgcggggga cgaatttccc ccccgacggt cccgtcatgc aaaagaaaac gatgggctgg
     4081 gaggcctcga ccgagcggct gtatccagaa gatggcgtgt tgaagggtga cattaaaatg
     4141 gccctgcggt tgaaagatgg tgggcgctac ttggcggact tcaaaaccac ctacaaagcc
     4201 aaaaaacccg tgcaaatgcc cggggcgtat aatgtcgacc gtaagctgga tattacgagc
     4261 cataacgaag attacacggt ggtggaacaa tacgaacgct cggagggtcg tcattccacc
     4321 ggggggatgg atgaattgta taagtgaaag cttatcgttt taattaagga ggtatctcca
     4381 tgagcggtca ccatcaccat caccacggcg gtagcgccat ggtgtctaag ggcgaagagc
     4441 tgatcaagga aaatatgcgt atgaaggtgg tcatggaagg ttcggtcaac ggccaccaat
     4501 tcaaatgcac aggtgaagga gaaggcagac cgtacgaggg agtgcaaacc atgaggatca
     4561 aagtcatcga gggaggaccc ctgccatttg cctttgacat tcttgccacg tcgttcatgt
     4621 atggcagccg tacctttatc aagtacccgg ccgacatccc tgatttcttt aaacagtcct
     4681 ttcctgaggg ttttacttgg gaaagagtta cgagatacga agatggtgga gtcgtcaccg
     4741 tcacgcagga caccagcctt gaggatggcg agctcgtcta caacgtcaag gtcagagggg
     4801 taaactttcc ctccaatggt cccgtgatgc agaagaagac caagggttgg gagcctaata
     4861 cagagatgat gtatccagca gatggtggtc tgagaggata cactgacatc gcactgaaag
     4921 ttgatggtgg tggccatctg cactgcaact tcgtgacaac ttacaggtca aaaaagaccg
     4981 tcgggaacat caagatgccc ggtgtccatg ccgttgatca ccgcctggaa aggatcgagg
     5041 agagtgacaa tgaaacctac gtagtgcaaa gagaagtggc agttgccaaa tacagcaacc
     5101 ttggtggtgg catggacgag ctgtacaagt aaaagcttat cgatgtcgac gtagttaact
     5161 agcgtacgat cgactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     5221 tttcgtttta tgccatcatg gccgcggtga tcagctagag ccgtgaacga cagggcgaac
     5281 gccagcccgc cgacggcgag ggttccgacc gctgcaactc ccggtgcaac cttgtcccgg
     5341 tctattctct tcactgcacc agctccaatc tggtgtgaat gcccctcgtc tgttcgcgca
     5401 ggcggggggc tctattcgtt tgtcagcatc gaaagtagcc agatcaggga tgcgttgcaa
     5461 ccg
//
