LOCUS       sequence-332743-        4064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis 
            Reporter Constructs for mWasabi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4064)
  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 4064)
  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::Pleft* mWasabi
FEATURES             Location/Qualifiers
     source          1..4064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             247..255
                     /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             271..288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             301..1014
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MSVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYD
                     GTNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMT
                     FEDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVL
                     KGDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEI
                     AVARNSTDGMDELYK"
     terminator      1067..1113
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1922..1941)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1933..2748
                     /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(2006..2025)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3079..3667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3568..3587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3897..3916
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3897..3915
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4032..4049
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccaatacgca aaccgcctct cccagatctg atatcgctag aggaaacagc tatgaccatg
       61 attacgccaa gcttgcatgc ctgcagctag ggcaccaatt tgcgattagg gcttgacagc
      121 cacccggcca gtagtgcatt cttgtgtcac cgcagcagca aggcggtagg cggatccgag
      181 aggatcgtgc cggtgccggt gaagagagat tcgccgcccg aaatgagcac gatccgcatg
      241 cttaattaag gaggtatctc catgagcggt caccatcacc atcaccacgg cggtagcgcc
      301 atgagcgtga gcaagggcga ggagaccaca atgggcgtaa tcaagcccga catgaagatc
      361 aagctgaaga tggagggcaa cgtgaatggc cacgccttcg tgatcgaggg cgagggcgag
      421 ggcaagccct acgacggcac caacaccatc aacctggagg tgaaggaggg agcccccctg
      481 cccttctcct acgacattct gaccaccgcg ttcagttacg gcaacagggc cttcaccaag
      541 taccccgacg acatccccaa ctacttcaag cagtccttcc ccgagggcta ctcttgggag
      601 cgcaccatga ccttcgagga caagggcatc gtgaaggtga agtccgacat ctccatggag
      661 gaggactcct tcatctacga gatacacctc aagggcgaga acttcccccc caacggcccc
      721 gtgatgcaga aggagaccac cggctgggac gcctccaccg agaggatgta cgtgcgcgac
      781 ggcgtgctga agggcgacgt caagatgaag ctgctgctgg agggcggcgg ccaccaccgc
      841 gttgacttca agaccatcta cagggccaag aaggcggtga agctgcccga ctatcacttt
      901 gtggaccacc gcatcgagat cctgaaccac gacaaggact acaacaaggt gaccgtttac
      961 gagatcgccg tggcccgcaa ctccaccgac ggcatggacg agctgtacaa gtaaaagctt
     1021 atcgatgtcg acgtagttaa ctagcgtacg atcgactgcc aggcatcaaa taaaacgaaa
     1081 ggctcagtcg aaagactggg cctttcgttt tatgccatca tggccgcggt gatcagctag
     1141 agccgtgaac gacagggcga acgccagccc gccgacggcg agggttccga ccgctgcaac
     1201 tcccggtgca accttgtccc ggtctattct cttcactgca ccagctccaa tctggtgtga
     1261 atgcccctcg tctgttcgcg caggcggggg gctctattcg tttgtcagca tcgaaagtag
     1321 ccagatcagg gatgcgttgc aaccgcgtat gcccaggtca gaagagtcgc acaagagttg
     1381 cagacccctg gaaagaaaaa tggccagagg gcgaaaacac cctctgacca gcggagcggg
     1441 cgacgggaat cgaacccgcg tagctagttt ggaagaatgg gtgtctgccg accacatata
     1501 tgggccggtc aagataggtt tttaccccct ctcggctgca tcctctaagt ggaaagaaat
     1561 tgcaggtcgt agaagcgcgt tgaagcctga gagttgcaca ggagttgcaa cccggtagcc
     1621 ttgttcacga cgagaggaga cctagggatc ctcccaagga cactgagtcc taaagagggg
     1681 ggtttcttgt cagtacgcga agaaccacgc ctggccgcga gcgccagcac cgccgctctg
     1741 tgcggagacc tgggcaccag ccccgccgcc gccaggagca ttgccgttcc cgccagctag
     1801 ccacctgacg tcgggggggg ggaaagccac gttgtgtctc aaaatctctg atgttacatt
     1861 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
     1921 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctcgaggcc gcgattaaat
     1981 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     2041 ggtgcgacaa tctatcgctt gtatgggaag ccccatgcgc cagagttgtt tctgaaacat
     2101 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     2161 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     2221 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca
     2281 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     2341 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     2401 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     2461 caagtctgga aagaaatgca taatcttttg ccattctcac cggattcagt cgtcactcat
     2521 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     2581 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     2641 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     2701 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt
     2761 aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgg ctttgttgaa
     2821 taaatcgaac ttttgctgag ttgaaggatc agatcacgca tcttcccgac aacgcagacc
     2881 gttccgtggc aaagcaaaag ttcaaaatca ccaactggtc cacctacaac aaagctctca
     2941 ccaaccgtgg ctccctcact ttctggctgg atgatggggc gattcaggcc tggtatgagt
     3001 cagcaacacc ttcttcacga ggcagacctc actagttcca tgagcgtcag accccgtaga
     3061 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3121 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3181 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3241 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3301 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3361 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3421 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3481 cgccacgctt cccgagggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3541 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3601 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3661 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3721 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3781 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc aacgcgtgcg gccgcggtac
     3841 cagatcttta aatctagacg atgataagcg gtcaaacatg agaattcgcg gccgcataat
     3901 acgactcact atagggatct taattaaggc gcctcatgtt ctttcctgcg ttatcccctg
     3961 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4021 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgc
//
