LOCUS       sequence-332739-        4049 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis 
            Reporter Constructs for mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4049)
  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 4049)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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* mScarlet
FEATURES             Location/Qualifiers
     source          1..4049
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..65
                     /label=L4440
                     /note="L4440 vector, forward primer"
     RBS             327..335
                     /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             351..368
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             381..1076
                     /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(514..532)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     782..801
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      1132..1178
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1987..2006)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1998..2813
                     /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(2071..2090)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3144..3732
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3633..3652
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3962..3981
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3962..3980
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
       61 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct cccagatctg atatcgctag
      121 aggaaacagc tatgaccatg attacgccaa gcttgcatgc ctgcagctag ggcaccaatt
      181 tgcgattagg gcttgacagc cacccggcca gtagtgcatt cttgtgtcac cgcagcagca
      241 aggcggtagg cggatccgag aggatcgtgc cggtgccggt gaagagagat tcgccgcccg
      301 aaatgagcac gatccgcatg cttaattaag gaggtatctc catgagcggt caccatcacc
      361 atcaccacgg cggtagcgcc atggtgagca agggcgaggc agtgatcaag gagttcatgc
      421 ggttcaaggt gcacatggag ggctccatga acggccacga gttcgagatc gagggcgagg
      481 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
      541 ccctgccctt ctcctgggac atcctgtccc ctcagttcat gtacggctcc agggccttca
      601 ccaagcaccc cgccgacatc cccgactact ataagcagtc cttccccgag ggcttcaagt
      661 gggagcgcgt gatgaacttc gaggacggcg gcgccgtgac cgtgacccag gacacctccc
      721 tggaggacgg caccctgatc tacaaggtga agctccgcgg caccaacttc cctcctgacg
      781 gccccgtaat gcagaagaag acaatgggct gggaagcgtc caccgagcgg ttgtaccccg
      841 aggacggcgt gctgaagggc gacattaaga tggccctgcg cctgaaggac ggcggccgct
      901 acctggcgga cttcaagacc acctacaagg ccaagaagcc cgtgcagatg cccggcgcct
      961 acaacgtcga ccgcaagttg gacatcacct cccacaacga ggactacacc gtggtggaac
     1021 agtacgaacg ctccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtgaa
     1081 agcttatcga tgtcgacgta gttaactagc gtacgatcga ctgccaggca tcaaataaaa
     1141 cgaaaggctc agtcgaaaga ctgggccttt cgttttatgc catcatggcc gcggtgatca
     1201 gctagagccg tgaacgacag ggcgaacgcc agcccgccga cggcgagggt tccgaccgct
     1261 gcaactcccg gtgcaacctt gtcccggtct attctcttca ctgcaccagc tccaatctgg
     1321 tgtgaatgcc cctcgtctgt tcgcgcaggc ggggggctct attcgtttgt cagcatcgaa
     1381 agtagccaga tcagggatgc gttgcaaccg cgtatgccca ggtcagaaga gtcgcacaag
     1441 agttgcagac ccctggaaag aaaaatggcc agagggcgaa aacaccctct gaccagcgga
     1501 gcgggcgacg ggaatcgaac ccgcgtagct agtttggaag aatgggtgtc tgccgaccac
     1561 atatatgggc cggtcaagat aggtttttac cccctctcgg ctgcatcctc taagtggaaa
     1621 gaaattgcag gtcgtagaag cgcgttgaag cctgagagtt gcacaggagt tgcaacccgg
     1681 tagccttgtt cacgacgaga ggagacctag ggatcctccc aaggacactg agtcctaaag
     1741 aggggggttt cttgtcagta cgcgaagaac cacgcctggc cgcgagcgcc agcaccgccg
     1801 ctctgtgcgg agacctgggc accagccccg ccgccgccag gagcattgcc gttcccgcca
     1861 gctagccacc tgacgtcggg gggggggaaa gccacgttgt gtctcaaaat ctctgatgtt
     1921 acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca
     1981 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctcg aggccgcgat
     2041 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     2101 aatcaggtgc gacaatctat cgcttgtatg ggaagcccca tgcgccagag ttgtttctga
     2161 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     2221 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     2281 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg
     2341 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     2401 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     2461 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     2521 ttgaacaagt ctggaaagaa atgcataatc ttttgccatt ctcaccggat tcagtcgtca
     2581 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     2641 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     2701 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     2761 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat
     2821 tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcggctttg
     2881 ttgaataaat cgaacttttg ctgagttgaa ggatcagatc acgcatcttc ccgacaacgc
     2941 agaccgttcc gtggcaaagc aaaagttcaa aatcaccaac tggtccacct acaacaaagc
     3001 tctcaccaac cgtggctccc tcactttctg gctggatgat ggggcgattc aggcctggta
     3061 tgagtcagca acaccttctt cacgaggcag acctcactag ttccatgagc gtcagacccc
     3121 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3181 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3241 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3301 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3361 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3421 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3481 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3541 gaaagcgcca cgcttcccga ggggagaaag gcggacaggt atccggtaag cggcagggtc
     3601 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3661 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3721 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3781 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3841 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcaacgc gtgcggccgc
     3901 ggtaccagat ctttaaatct agacgatgat aagcggtcaa acatgagaat tcgcggccgc
     3961 ataatacgac tcactatagg gatcttaatt aaggcgcctc atgttctttc ctgcgttatc
     4021 ccctgattct gtggataacc gtattaccg
//
