LOCUS       sequence-333250-        4677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis 
            Reporter Constructs for double blue.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4677)
  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 4677)
  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 double blue
FEATURES             Location/Qualifiers
     source          1..4677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(791..810)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             802..1617
                     /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(875..894)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1948..2536
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2437..2456
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             3036..3044
                     /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             3060..3077
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3090..3794
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MGSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     RBS             3808..3816
                     /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             3832..3849
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4612..4658
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tcagctagag ccgtgaacga cagggcgaac gccagcccgc cgacggcgag ggttccgacc
       61 gctgcaactc ccggtgcaac cttgtcccgg tctattctct tcactgcacc agctccaatc
      121 tggtgtgaat gcccctcgtc tgttcgcgca ggcggggggc tctattcgtt tgtcagcatc
      181 gaaagtagcc agatcaggga tgcgttgcaa ccgcgtatgc ccaggtcaga agagtcgcac
      241 aagagttgca gacccctgga aagaaaaatg gccagagggc gaaaacaccc tctgaccagc
      301 ggagcgggcg acgggaatcg aacccgcgta gctagtttgg aagaatgggt gtctgccgac
      361 cacatatatg ggccggtcaa gataggtttt taccccctct cggctgcatc ctctaagtgg
      421 aaagaaattg caggtcgtag aagcgcgttg aagcctgaga gttgcacagg agttgcaacc
      481 cggtagcctt gttcacgacg agaggagacc tagggatcct cccaaggaca ctgagtccta
      541 aagagggggg tttcttgtca gtacgcgaag aaccacgcct ggccgcgagc gccagcaccg
      601 ccgctctgtg cggagacctg ggcaccagcc ccgccgccgc caggagcatt gccgttcccg
      661 ccagctagcc acctgacgtc gggggggggg gagagccacg ttgtgtctca aaatctctga
      721 tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata
      781 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg ctcgaggccg
      841 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
      901 gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc cccatgcgcc agagttgttt
      961 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     1021 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     1081 gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat
     1141 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     1201 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     1261 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     1321 cctgttgaac aagtctggaa agaaatgcat aatcttttgc cattctcacc ggattcagtc
     1381 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     1441 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     1501 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     1561 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca
     1621 gaattggtta attggttgta acactggcag agcattacgc tgacttgacg ggacggcggc
     1681 tttgttgaat aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttcccgaca
     1741 acgcagaccg ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca
     1801 aagctctcac caaccgtggc tccctcactt tctggctgga tgatggggcg attcaggcct
     1861 ggtatgagtc agcaacacct tcttcacgag gcagacctca ctagttccat gagcgtcaga
     1921 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1981 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2041 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2101 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2161 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2221 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2281 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2341 atgagaaagc gccacgcttc ccgaggggag aaaggcggac aggtatccgg taagcggcag
     2401 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2461 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2521 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2581 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2641 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca acgcgtgcgg
     2701 ccgcggtacc agatctttaa atctagagga tcgtcggcac cgtcacggcc gtgggaggcg
     2761 gcacgatccg cgacgtgatg atcggccgca tccccacggt gctgcgcagt gagctctacg
     2821 ccatcccggc gttgatctgt gcgttcgcac gcacaggccc ggtgtgagaa gggtctctga
     2881 cgagcgggag aacccacccg gggtgggcga gtttgtcctg cgtgtgctcg gtcgagtagg
     2941 ctctgggagt acccgtgtgt acgaccagca cggcatacat catttcgacg ccgagagatt
     3001 cgccgcccga aatgagcacg atccgcatgc ttaattaagg aggtatctcc atgagcggtc
     3061 accatcacca tcaccacggc ggtagcgcca tgggcagcga gctgattaag gagaacatgc
     3121 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg
     3181 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc
     3241 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca
     3301 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat
     3361 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc
     3421 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg
     3481 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg
     3541 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc
     3601 tgatcgcaaa cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc
     3661 ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct
     3721 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc
     3781 acaagctcaa ttaaaagctt gcttaattaa ggaggtatct ccatgagcgg tcaccatcac
     3841 catcaccacg gcggtagcgc catgagctcg gagctgatca aggagaacat gcacatgaag
     3901 ctgtacatgg agggcaccgt ggacaaccac cacttcaagt gcacctcgga gggcgagggc
     3961 aagccgtacg agggcaccca gaccatgcgc atcaaggtgg tggagggcgg cccgctgccg
     4021 ttcgccttcg acatcctggc cacctcgttc ctgtacggct cgaagacctt catcaaccac
     4081 acccagggca tcccggactt cttcaagcag tcgttcccgg agggcttcac ctgggagcgc
     4141 gtgaccacct acgaggacgg cggcgtgctg accgccaccc aggacacctc gctgcaggac
     4201 ggctgcctga tctacaacgt gaagatccgc ggcgtgaact tcacctcgaa cggcccggtg
     4261 atgcagaaga agaccctggg ctgggaggcc ttcaccgaga ccctgtaccc ggccgacggc
     4321 ggcctggagg gccgcaacga catggccctg aagctggtgg gcggctcgca cctgatcgcc
     4381 aacgccaaga ccacctaccg ctcgaagaag ccggccaaga acctgaagat gccgggcgtg
     4441 tactacgtgg actaccgcct ggagcgcatc aaggaggcca acaacgagac ctacgtggag
     4501 cagcacgagg tggccgtggc ccgctactgc gacctgccgt cgaagctggg ccacaagctg
     4561 aactaatcga tgtcgacgta gttaactagc gtacgatcga ctgccaggca tcaaataaaa
     4621 cgaaaggctc agtcgaaaga ctgggccttt cgttttatgc catcatggcc gcggtga
//
