LOCUS       sequence-332742-        4710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis 
            Reporter Constructs for double green.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4710)
  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 4710)
  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 green
FEATURES             Location/Qualifiers
     source          1..4710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(761..780)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             772..1587
                     /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(845..864)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1918..2506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2407..2426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             3006..3014
                     /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             3030..3047
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3060..3773
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MSVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYD
                     GTNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMT
                     FEDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVL
                     KGDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEI
                     AVARNSTDGMDELYK"
     RBS             3794..3802
                     /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             3818..3835
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4615..4661
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gccagcccgc cgacggcgag ggttccgacc gctgcaactc ccggtgcaac cttgtcccgg
       61 tctattctct tcactgcacc agctccaatc tggtgtgaat gcccctcgtc tgttcgcgca
      121 ggcggggggc tctattcgtt tgtcagcatc gaaagtagcc agatcaggga tgcgttgcaa
      181 ccgcgtatgc ccaggtcaga agagtcgcac aagagttgca gacccctgga aagaaaaatg
      241 gccagagggc gaaaacaccc tctgaccagc ggagcgggcg acgggaatcg aacccgcgta
      301 gctagtttgg aagaatgggt gtctgccgac cacatatatg ggccggtcaa gataggtttt
      361 taccccctct cggctgcatc ctctaagtgg aaagaaattg caggtcgtag aagcgcgttg
      421 aagcctgaga gttgcacagg agttgcaacc cggtagcctt gttcacgacg agaggagacc
      481 tagggatcct cccaaggaca ctgagtccta aagagggggg tttcttgtca gtacgcgaag
      541 aaccacgcct ggccgcgagc gccagcaccg ccgctctgtg cggagacctg ggcaccagcc
      601 ccgccgccgc caggagcatt gccgttcccg ccagctagcc acctgacgtc gggggggggg
      661 gagagccacg ttgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
      721 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
      781 attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta
      841 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg
      901 tatgggaagc cccatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
      961 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     1021 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     1081 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     1141 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     1201 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     1261 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     1321 aatcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     1381 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     1441 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     1501 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     1561 catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag
     1621 agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact tttgctgagt
     1681 tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca aagcaaaagt
     1741 tcaaaatcac caactggtcc acctacaaca aagctctcac caaccgtggc tccctcactt
     1801 tctggctgga tgatggggcg attcaggcct ggtatgagtc agcaacacct tcttcacgag
     1861 gcagacctca ctagttccat gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1921 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1981 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2041 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     2101 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2161 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2221 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2281 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaggggag
     2341 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2401 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2461 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2521 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2581 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2641 cagccgaacg accgagcgca acgcgtgcgg ccgcggtacc agatctttaa atctagagga
     2701 tcgtcggcac cgtcacggcc gtgggaggcg gcacgatccg cgacgtgatg atcggccgca
     2761 tccccacggt gctgcgcagt gagctctacg ccatcccggc gttgatctgt gcgttcgcac
     2821 gcacaggccc ggtgtgagaa gggtctctga cgagcgggag aacccacccg gggtgggcga
     2881 gtttgtcctg cgtgtgctcg gtcgagtagg ctctgggagt acccgtgtgt acgaccagca
     2941 cggcatacat catttcgacg ccgagagatt cgccgcccga aatgagcacg atccgcatgc
     3001 ttaattaagg aggtatctcc atgagcggtc accatcacca tcaccacggc ggtagcgcca
     3061 tgagcgtgag caagggcgag gagaccacaa tgggcgtaat caagcccgac atgaagatca
     3121 agctgaagat ggagggcaac gtgaatggcc acgccttcgt gatcgagggc gagggcgagg
     3181 gcaagcccta cgacggcacc aacaccatca acctggaggt gaaggaggga gcccccctgc
     3241 ccttctccta cgacattctg accaccgcgt tcagttacgg caacagggcc ttcaccaagt
     3301 accccgacga catccccaac tacttcaagc agtccttccc cgagggctac tcttgggagc
     3361 gcaccatgac cttcgaggac aagggcatcg tgaaggtgaa gtccgacatc tccatggagg
     3421 aggactcctt catctacgag atacacctca agggcgagaa cttccccccc aacggccccg
     3481 tgatgcagaa ggagaccacc ggctgggacg cctccaccga gaggatgtac gtgcgcgacg
     3541 gcgtgctgaa gggcgacgtc aagatgaagc tgctgctgga gggcggcggc caccaccgcg
     3601 ttgacttcaa gaccatctac agggccaaga aggcggtgaa gctgcccgac tatcactttg
     3661 tggaccaccg catcgagatc ctgaaccacg acaaggacta caacaaggtg accgtttacg
     3721 agatcgccgt ggcccgcaac tccaccgacg gcatggacga gctgtacaag taaaagcttt
     3781 ccgcatgctt aattaaggag gtatctccat gagcggtcac catcaccatc accacggcgg
     3841 tagcgccatg ggcagcgtga gcaagggcga ggagaccaca atgggcgtga tcaagccgga
     3901 catgaagatc aagctgaaga tggagggcaa cgtgaacggc cacgccttcg tgatcgaggg
     3961 cgagggcgag ggcaagccgt acgacggcac caacaccatc aacctggagg tgaaggaggg
     4021 cgccccgctg ccgttctcgt acgacatcct gaccaccgcc ttctcgtacg gcaaccgcgc
     4081 cttcaccaag tacccggacg acatcccgaa ctacttcaag cagtcgttcc cggagggcta
     4141 ctcgtgggag cgcaccatga ccttcgagga caagggcatc gtgaaggtga agtcggacat
     4201 ctcgatggag gaggactcgt tcatctacga gatccacctg aagggcgaga acttcccgcc
     4261 gaacggcccg gtgatgcaga aggagaccac cggctgggac gcctcgaccg agcgcatgta
     4321 cgtgcgcgac ggcgtgctga agggcgacgt gaagatgaag ctgctgctgg agggcggcgg
     4381 ccaccaccgc gtggacttca agaccatcta ccgcgccaag aaggccgtga agctgccgga
     4441 ctaccacttc gtggaccacc gcatcgagat cctgaaccac gacaaggact acaacaaggt
     4501 gaccgtgtac gagatcgccg tggcccgcaa ctcgaccgac ggcatggacg agctgtacaa
     4561 gtaagcttat cgatgtcgac gtagttaact agcgtacgat cgactgccag gcatcaaata
     4621 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tgccatcatg gccgcggtga
     4681 tcagctagag ccgtgaacga cagggcgaac
//
