LOCUS       sequence-455970-        9914 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kanamycin resistant L5-integrative vector for CRISPR interference in
            M.tuberculosis with the constitutive expression of tdTomato under 
            the control of the psmyc promotor (psmyc::tdTomato).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9914)
  AUTHORS   Laudouze J, Point V, Achache W, Crauste C, Canaan S, Santucci P
  TITLE     Fluorescence-based CRISPR interference system for controlled genetic
            repression and live single-cell imaging in mycobacteria.
  JOURNAL   FEBS Lett. 2025 Feb;599(4):488-501. doi: 10.1002/1873-3468.15071. 
            Epub 2024 Dec 1.
  PUBMED    39618159
REFERENCE   2  (bases 1 to 9914)
  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"; doi: 
            "10.1002/1873-3468.15071"; journalName: "FEBS Lett"; date: 
            "2025-02"; volume: "599"; issue: "4"; pages: "488-501"
COMMENT     Sequence Label: pJL34
FEATURES             Location/Qualifiers
     source          1..9914
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    330..348
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    360..378
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      3858..3885
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4726..5427)
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(4809..4832)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(5007..5026)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5276..5294
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7934..7953)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7945..8760
                     /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(8018..8037)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(9473..9914,1..147))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9553..9572)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
       61 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
      121 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
      181 gctcatggaa caataaacga aaggctcagt cgaaagactg ggcctttcgt ttatctgttg
      241 tttgaaaaaa aaaagccccg cgattgcggg gctttttttt ttcatatgtc tgaccaggga
      301 aaatagccct ctgacctggg gatttgactt ccctatcagt gatagagata atctgggagt
      361 ccctatcagt gatagagaac gcggtatcga tatgtcggac ctggtgctgg gcctggccat
      421 cggcatcggc tcggtgggcg tgggcatcct gaacaaggtg accggcgaga tcatccacaa
      481 gaactcgcgc atcttcccgg ccgcccaggc cgagaacaac ctggtgcgcc gcaccaaccg
      541 ccagggccgc cgcctggccc gccgcaagaa gcaccgccgc gtgcgcctga accgcctgtt
      601 cgaggagtcg ggcctgatca ccgacttcac caagatctcg atcaacctga acccgtacca
      661 gctgcgcgtg aagggcctga ccgacgagct gtcgaacgag gagctgttca tcgccctgaa
      721 gaacatggtg aagcaccgcg gcatctcgta cctggacgac gcctcggacg acggcaactc
      781 gtcggtgggc gactacgccc agatcgtgaa ggagaactcg aagcagctgg agaccaagac
      841 cccgggccag atccagctgg agcgctacca gacctacggc cagctgcgcg gcgacttcac
      901 cgtggagaag gacggcaaga agcaccgcct gatcaacgtg ttcccgacct cggcctaccg
      961 ctcggaggcc ctgcgcatcc tgcagaccca gcaggagttc aacccgcaga tcaccgacga
     1021 gttcatcaac cgctacctgg agatcctgac cggcaagcgc aagtactacc acggcccggg
     1081 caacgagaag tcgcgcaccg actacggccg ctaccgcacc tcgggcgaga ccctggacaa
     1141 catcttcggc atcctgatcg gcaagtgcac cttctacccg gacgagttcc gcgccgccaa
     1201 ggcctcgtac accgcccagg agttcaacct gctgaacgac ctgaacaacc tgaccgtgcc
     1261 gaccgagacc aagaagctgt cgaaggagca gaagaaccag atcatcaact acgtgaagaa
     1321 cgagaaggcc atgggcccgg ccaagctgtt caagtacatc gccaagctgc tgtcgtgcga
     1381 cgtggccgac atcaagggct accgcatcga caagtcgggc aaggccgaga tccacacctt
     1441 cgaggcctac cgcaagatga agaccctgga gaccctggac atcgagcaga tggaccgcga
     1501 gaccctggac aagctggcct acgtgctgac cctgaacacc gagcgcgagg gcatccagga
     1561 ggccctggag cacgagttcg ccgacggctc gttctcgcag aagcaggtgg acgagctggt
     1621 gcagttccgc aaggccaact cgtcgatctt cggcaagggc tggcacaact tctcggtgaa
     1681 gctgatgatg gagctgatcc cggagctgta cgagacctcg gaggagcaga tgaccatcct
     1741 gacccgcctg ggcaagcaga agaccacctc gtcgtcgaac aagaccaagt acatcgacga
     1801 gaagctgctg accgaggaga tctacaaccc ggtggtggcc aagtcggtgc gccaggccat
     1861 caagatcgtg aacgccgcca tcaaggagta cggcgacttc gacaacatcg tgatcgagat
     1921 ggcccgcgag accaacgagg acgacgagaa gaaggccatc cagaagatcc agaaggccaa
     1981 caaggacgag aaggacgccg ccatgctgaa ggccgccaac cagtacaacg gcaaggccga
     2041 gctgccgcac tcggtgttcc acggccacaa gcagctggcc accaagatcc gcctgtggca
     2101 ccagcagggc gagcgctgcc tgtacaccgg caagaccatc tcgatccacg acctgatcaa
     2161 caactcgaac cagttcgagg tggacgccat cctgccgctg tcgatcacct tcgacgactc
     2221 gctggccaac aaggtgctgg tgtacgccac cgccaaccag gagaagggcc agcgcacccc
     2281 gtaccaggcc ctggactcga tggacgacgc ctggtcgttc cgcgagctga aggccttcgt
     2341 gcgcgagtcg aagaccctgt cgaacaagaa gaaggagtac ctgctgaccg aggaggacat
     2401 ctcgaagttc gacgtgcgca agaagttcat cgagcgcaac ctggtggaca cccgctacgc
     2461 ctcgcgcgtg gtgctgaacg ccctgcagga gcacttccgc gcccacaaga tcgacaccaa
     2521 ggtgtcggtg gtgcgcggcc agttcacctc gcagctgcgc cgccactggg gcatcgagaa
     2581 gacccgcgac acctaccacc accacgccgt ggacgccctg atcatcgccg cctcgtcgca
     2641 gctgaacctg tggaagaagc agaagaacac cctggtgtcg tactcggagg accagctgct
     2701 ggacatcgag accggcgagc tgatctcgga cgacgagtac aaggagtcgg tgttcaaggc
     2761 cccgtaccag cacttcgtgg acaccctgaa gtcgaaggag ttcgaggact cgatcctgtt
     2821 ctcgtaccag gtggactcga agttcaaccg caagatctcg gacgccacca tctacgccac
     2881 ccgccaggcc aaggtgggca aggacaaggc cgacgagacc tacgtgctgg gcaagatcaa
     2941 ggacatctac acccaggacg gctacgacgc cttcatgaag atctacaaga aggacaagtc
     3001 gaagttcctg atgtaccgcc acgacccgca gaccttcgag aaggtgatcg agccgatcct
     3061 ggagaactac ccgaacaagc agatcaacga caagggcaag gaggtgccgt gcaacccgtt
     3121 cctgaagtac aaggaggagc acggctacat ccgcaagtac tcgaagaagg gcaacggccc
     3181 ggagatcaag tcgctgaagt actacgactc gaagctgggc aaccacatcg acatcacccc
     3241 gaaggactcg aacaacaagg tggtgctgca gtcggtgtcg ccgtggcgcg ccgacgtgta
     3301 cttcaacaag accaccggca agtacgagat cctgggcctg aagtacgccg acctgcagtt
     3361 cgacaagggc accggcacct acaagatctc gcaggagaag tacaacgaca tcaagaagaa
     3421 ggagggcgtg gactcggact cggagttcaa gttcaccctg tacaagaacg acctgctgct
     3481 ggtgaaggac accgagacca aggagcagca gctgttccgc ttcctgtcgc gcaccatgcc
     3541 gaagcagaag cactacgtgg agctgaagcc gtacgacaag cagaagttcg agggcggcga
     3601 ggccctgatc aaggtgctgg gcaacgtggc caactcgggc cagtgcaaga agggcctggg
     3661 caagtcgaac atctcgatct acaaggtgcg caccgacgtg ctgggcaacc agcacatcat
     3721 caagaacgag ggcgacaagc cgaagctgga cttctaaata ctctggccat ttttctttac
     3781 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca acgcgtgcgg
     3841 ccgctgatta gctaagcaga aggccatcct gacggatggc ctttttctag aaaaaaaaaa
     3901 agcgccgcaa ctgcggcgct tttttttttt taattaagtg atagattgcg atgcgtgtgc
     3961 tcggttgata taatctgtca gatcaggagg acttcgcatg tcgcggttgg ataagtccaa
     4021 ggtcatcaac tcggcgctgg agctgctgaa cgaggtcggt atcgagggac tgaccacgcg
     4081 caagttggcc caaaagctcg gcgtggaaca accgaccctc tactggcacg tgaagaacaa
     4141 gcgcgcgctg ctggatgcgt tggccatcga gatgctggat cgccatcaca cgcacttttg
     4201 ccccctggag ggcgagagct ggcaggattt tctgcggaac aacgcgaagt cgttccggtg
     4261 cgccctcctg agccatcggg atggcgccaa agtccacttg ggcacccgcc cgaccgagaa
     4321 gcaatacgag accctggaga accagctggc cttcctctgt caacagggtt tttcgttgga
     4381 gaacgccctg tatgccctgt ccgccgtggg acacttcacc ttgggctgcg tgttggagga
     4441 tcaggaacat caggtggcga aagaggaacg ggagaccccc acgacggatt cgatgccccc
     4501 gttgttgcgc caggcgatcg aactgtttga ccaccagggc gccgaacccg cctttttgtt
     4561 cgggctggag ctgatcatct gcgggctgga aaagcaactg aagtgcgaaa gcggttcgtg
     4621 ataacgccga aaaccccgct tcggcggggt tttgccgcgg tctgcgttgt cgggaagatt
     4681 taaaataaaa aaggggacct ctagggtccc caattaaagc ttctacttgt acagctcgtc
     4741 catgccgtac aggaacaggt ggtggcggcc ctcggagcgc tcgtactgtt ccacgatggt
     4801 gtagtcctcg ttgtgggagg tgatgtccag cttggtgtcc acgtagtagt agccgggcag
     4861 ttgcacgggc ttcttggcca tgtagatggt cttgaactcc accaggtagt ggccgccgtc
     4921 cttcagcttc agggcctggt ggatctcgcc cttcagcacg ccgtcgcggg ggtacaggcg
     4981 ctcggtggag gcctcccagc ccatggtctt cttctgcatt acggggccgt cgggggggaa
     5041 gttggtgccg cgcatcttca ccttgtagat cagcgtgccg tcctgcaggg aggagtcctg
     5101 ggtcacggtc accagaccgc cgtcctcgaa gttcatcacg cgctcccact tgaagccctc
     5161 ggggaaggac agcttcttgt aatcggggat gtcggcgggg tgcttcacgt acgccttgga
     5221 gccgtacatg aactgggggg acaggatgtc ccaggcgaag ggcagggggc cgcccttggt
     5281 caccttcagc ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat
     5341 ctcgaactcg tggccgttca tggagccctc catgcgcacc ttgaagcgca tgaactcttt
     5401 gatgacctcc tcgcccttgc tcaccatacc ggtgcctcct taattaagca tgcggatcgt
     5461 gctcatttcg ggcggcgaat ctctcggcgt cgaaatgatg tatgccgtgc tggtcgtaca
     5521 cacgggtact cccagagcct actcgaccga gcacacgcag gacaaactcg cccaccccgg
     5581 gtgggttctc ccgctcgtca gagacccttc tcacaccggg cctgtgcgtg cgaacgcaca
     5641 gatcaacgcc gggatggcgt agagctcact gcgcagcacc gtggggatgc ggccgatcat
     5701 cacgtcgcgg atcgtgccgc ctcccacggc cgtgacggtg ccgacgatcc aatatttcta
     5761 gagtttaaac agtattaaac gcgaattctt ataaagcttc tacttgtaca gctcgtcaga
     5821 gacccttctc acaccgggcc tgtgcgtgcg aacgcacaga tcaacgccgg gatggcgtag
     5881 agctcactgc gcagcaccgt ggggatgcgg ccgatcatca cgtcgcggat cgtgccgcct
     5941 cccacggccg tgacggtgcc gacgatccaa tatttctaga gtttaaacag tattaaacgc
     6001 gaattctata ctagtattaa aaaaaaaagc cccgcgattg cggggctttt ttttttcgac
     6061 ggcgagggtt ccgaccgctg caactcccgg tgcaaccttg tcccggtcta ttctcttcac
     6121 tgcaccagct ccaatctggt gtgaatgccc ctcgtctgtt cgcgcaggcg gggggctcta
     6181 ttcgtttgtc agcatcgaaa gtagccagat cagggatgcg ttgcaaccgc gtatgcccag
     6241 gtcagaagag tcgcacaaga gttgcagacc cctggaaaga aaaatggcca gagggcgaaa
     6301 acaccctctg accagcggag cgggcgacgg gaatcgaacc cgcgtagcta gtttggaaga
     6361 atgggtgtct gccgaccaca tatatgggcc ggtcaagata ggtttttacc ccctctcggc
     6421 tgcatcctct aagtggaaag aaattgcagg tcgtagaagc gcgttgaagc ctgagagttg
     6481 cacaggagtt gcaacccggt agccttgttc acgacgagag gagacctagt tggcacgtcg
     6541 cggatgggga tcgctgaaga ctcagcgcag cgggaggatc caagcctcat acgtcaaccc
     6601 gcaggacggt gtgaggtact acgcgctgca gacctacgac aacaagatgg acgccgaagc
     6661 ctggctcgcg ggcgagaagc ggctcatcga gatggagacc tggacccctc cacaggaccg
     6721 ggcgaagaag gcagccgcca gcgccatcac gctggaggag tacacccgga agtggctcgt
     6781 ggagcgcgac ctcgcagacg gcaccaggga tctgtacagc gggcacgcgg agcgccgcat
     6841 ctacccggtg ctaggtgaag tggcggtcac agagatgacg ccagctctgg tgcgtgcgtg
     6901 gtgggccggg atgggtagga agcacccgac tgcccgccgg catgcctaca acgtcctccg
     6961 ggcggtgatg aacacagcgg tcgaggacaa gctgatcgca gagaacccgt gccggatcga
     7021 gcagaaggca gccgatgagc gcgacgtaga ggcgctgacg cctgaggagc tggacatcgt
     7081 cgccgctgag atcttcgtgc actaccggat cgcggcatac atcctggcgt ggacgagcct
     7141 ccggttcgga gagctgatcg agcttcgccg caaggacatc gtggacgacg gcatgacgat
     7201 gaagctccgg gtgcgccgtg gcgcttcccg cgtggggaac aagatcgtcg ttggcaacgc
     7261 caagaccgtc cggtcgaagc gtcctgtgac ggttccgcct cacgtcgcgg agatgatccg
     7321 agcgcacatg aaggaccgta cgaagatgaa caagggcccc gaggcattcc tggtgaccac
     7381 gacgcagggc aaccggctgt cgaagtccgc gttcaccaag tcgctgaagc gtggctacgc
     7441 caagatcggt cggccggaac tccgcatcca cgacctccgc gctgtcggcg ctacgttcgc
     7501 cgctcaggca ggtgcgacga ccaaggagct gatggcccgc ctcggtcaca cgactcctag
     7561 gatggcgatg aagtaccaga tggcgtctga ggcccgcgac gaggctatcg ctgaggcgat
     7621 gtccaagctg gccaagacct cctgaaacgc aaaaagcccc cctcccaagg acactgagtc
     7681 ctaaagaggg gggtttcttg tcagtacgcg aagaaccacg cctggccgcg agcgccagca
     7741 ccgccgctct gtgcggagac ctgggcacca gccccgccgc cgccaggagc attgccgttc
     7801 ccgccagcta gccacctgac gtcggggggg ggggaaagcc acgttgtgtc tcaaaatctc
     7861 tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac
     7921 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctcgagg
     7981 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     8041 gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agccccatgc gccagagttg
     8101 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     8161 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     8221 gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa
     8281 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     8341 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgcct cgctcaggcg
     8401 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     8461 tggcctgttg aacaagtctg gaaagaaatg cataatcttt tgccattctc accggattca
     8521 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     8581 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     8641 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     8701 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     8761 tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc
     8821 ggctttgttg aataaatcga acttttgctg agttgaagga tcagatcacg catcttcccg
     8881 acaacgcaga ccgttccgtg gcaaagcaaa agttcaaaat caccaactgg tccacctaca
     8941 acaaagctct caccaaccgt ggctccctca cgatatcaat aaacgaaagg ctcagtcgaa
     9001 agactgggcc tttcgtttat ctgttgtttg aaaaaaaaaa gcgccgcaac tgcggcgctt
     9061 ttttttttga attctctgac cagggaaaat agccctctga cctggggatt tgcgatccct
     9121 atcagtgata gatataatct gggaggagac gattaatgcg tctcggtttt tgtactcgaa
     9181 agaagctaca aagataaggc ttcatgccga aatcaacacc ctgtcatttt atggcagggt
     9241 gttttttttt tgtcgacttg gggaccctag aggtcccctt ttttattttt ttggatgaag
     9301 agcattatca atggctcttc acaggaagct taaaaaaaaa agccccgcga ttgcggggct
     9361 tttttttttt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     9421 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     9481 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     9541 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     9601 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccctcggga agcgtggcgc
     9661 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     9721 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     9781 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     9841 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     9901 gctacactag aagg
//
