LOCUS       sequence-455968-        9710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kanamycin resistant L5-integrative vector for CRISPR interference in
            M.smegmatis 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 9710)
  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 9710)
  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: pJL32
FEATURES             Location/Qualifiers
     source          1..9710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1436..2024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1516..1535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2207..2225
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2237..2255
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      5735..5762
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             6965..7666
                     /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(7098..7116)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7366..7385
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7560..7583
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(9607..9626)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(9618..9710,1..723)
                     /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(9691..9710)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccca tgcgccagag
       61 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
      121 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
      181 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
      241 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
      301 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg cctcgctcag
      361 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
      421 ggctggcctg ttgaacaagt ctggaaagaa atgcataatc ttttgccatt ctcaccggat
      481 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
      541 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
      601 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
      661 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
      721 taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac
      781 ggcggctttg ttgaataaat cgaacttttg ctgagttgaa ggatcagatc acgcatcttc
      841 ccgacaacgc agaccgttcc gtggcaaagc aaaagttcaa aatcaccaac tggtccacct
      901 acaacaaagc tctcaccaac cgtggctccc tcacgatatc aataaacgaa aggctcagtc
      961 gaaagactgg gcctttcgtt tatctgttgt ttgaaaaaaa aaagcgccgc aactgcggcg
     1021 cttttttttt tgaattctct gaccagggaa aatagccctc tgacctgggg atttgcgatc
     1081 cctatcagtg atagatataa tctgggagga gacgattaat gcgtctcggt ttttgtactc
     1141 gaaagaagct acaaagataa ggcttcatgc cgaaatcaac accctgtcat tttatggcag
     1201 ggtgtttttt ttttgtcgac ttggggaccc tagaggtccc cttttttatt tttttggatg
     1261 aagagcatta tcaatggctc ttcacaggaa gcttaaaaaa aaaagccccg cgattgcggg
     1321 gctttttttt tttaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1381 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1441 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     1501 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1561 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccctcg ggaagcgtgg
     1621 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1681 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1741 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1801 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1861 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1921 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1981 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2041 tttctacggg gtctgacgct catggaacaa taaacgaaag gctcagtcga aagactgggc
     2101 ctttcgttta tctgttgttt gaaaaaaaaa agccccgcga ttgcggggct tttttttttc
     2161 atatgtctga ccagggaaaa tagccctctg acctggggat ttgacttccc tatcagtgat
     2221 agagataatc tgggagtccc tatcagtgat agagaaggcg ctatcgatat gtcggacctg
     2281 gtgctgggcc tggccatcgg catcggctcg gtgggcgtgg gcatcctgaa caaggtgacc
     2341 ggcgagatca tccacaagaa ctcgcgcatc ttcccggccg cccaggccga gaacaacctg
     2401 gtgcgccgca ccaaccgcca gggccgccgc ctggcccgcc gcaagaagca ccgccgcgtg
     2461 cgcctgaacc gcctgttcga ggagtcgggc ctgatcaccg acttcaccaa gatctcgatc
     2521 aacctgaacc cgtaccagct gcgcgtgaag ggcctgaccg acgagctgtc gaacgaggag
     2581 ctgttcatcg ccctgaagaa catggtgaag caccgcggca tctcgtacct ggacgacgcc
     2641 tcggacgacg gcaactcgtc ggtgggcgac tacgcccaga tcgtgaagga gaactcgaag
     2701 cagctggaga ccaagacccc gggccagatc cagctggagc gctaccagac ctacggccag
     2761 ctgcgcggcg acttcaccgt ggagaaggac ggcaagaagc accgcctgat caacgtgttc
     2821 ccgacctcgg cctaccgctc ggaggccctg cgcatcctgc agacccagca ggagttcaac
     2881 ccgcagatca ccgacgagtt catcaaccgc tacctggaga tcctgaccgg caagcgcaag
     2941 tactaccacg gcccgggcaa cgagaagtcg cgcaccgact acggccgcta ccgcacctcg
     3001 ggcgagaccc tggacaacat cttcggcatc ctgatcggca agtgcacctt ctacccggac
     3061 gagttccgcg ccgccaaggc ctcgtacacc gcccaggagt tcaacctgct gaacgacctg
     3121 aacaacctga ccgtgccgac cgagaccaag aagctgtcga aggagcagaa gaaccagatc
     3181 atcaactacg tgaagaacga gaaggccatg ggcccggcca agctgttcaa gtacatcgcc
     3241 aagctgctgt cgtgcgacgt ggccgacatc aagggctacc gcatcgacaa gtcgggcaag
     3301 gccgagatcc acaccttcga ggcctaccgc aagatgaaga ccctggagac cctggacatc
     3361 gagcagatgg accgcgagac cctggacaag ctggcctacg tgctgaccct gaacaccgag
     3421 cgcgagggca tccaggaggc cctggagcac gagttcgccg acggctcgtt ctcgcagaag
     3481 caggtggacg agctggtgca gttccgcaag gccaactcgt cgatcttcgg caagggctgg
     3541 cacaacttct cggtgaagct gatgatggag ctgatcccgg agctgtacga gacctcggag
     3601 gagcagatga ccatcctgac ccgcctgggc aagcagaaga ccacctcgtc gtcgaacaag
     3661 accaagtaca tcgacgagaa gctgctgacc gaggagatct acaacccggt ggtggccaag
     3721 tcggtgcgcc aggccatcaa gatcgtgaac gccgccatca aggagtacgg cgacttcgac
     3781 aacatcgtga tcgagatggc ccgcgagacc aacgaggacg acgagaagaa ggccatccag
     3841 aagatccaga aggccaacaa ggacgagaag gacgccgcca tgctgaaggc cgccaaccag
     3901 tacaacggca aggccgagct gccgcactcg gtgttccacg gccacaagca gctggccacc
     3961 aagatccgcc tgtggcacca gcagggcgag cgctgcctgt acaccggcaa gaccatctcg
     4021 atccacgacc tgatcaacaa ctcgaaccag ttcgaggtgg acgccatcct gccgctgtcg
     4081 atcaccttcg acgactcgct ggccaacaag gtgctggtgt acgccaccgc caaccaggag
     4141 aagggccagc gcaccccgta ccaggccctg gactcgatgg acgacgcctg gtcgttccgc
     4201 gagctgaagg ccttcgtgcg cgagtcgaag accctgtcga acaagaagaa ggagtacctg
     4261 ctgaccgagg aggacatctc gaagttcgac gtgcgcaaga agttcatcga gcgcaacctg
     4321 gtggacaccc gctacgcctc gcgcgtggtg ctgaacgccc tgcaggagca cttccgcgcc
     4381 cacaagatcg acaccaaggt gtcggtggtg cgcggccagt tcacctcgca gctgcgccgc
     4441 cactggggca tcgagaagac ccgcgacacc taccaccacc acgccgtgga cgccctgatc
     4501 atcgccgcct cgtcgcagct gaacctgtgg aagaagcaga agaacaccct ggtgtcgtac
     4561 tcggaggacc agctgctgga catcgagacc ggcgagctga tctcggacga cgagtacaag
     4621 gagtcggtgt tcaaggcccc gtaccagcac ttcgtggaca ccctgaagtc gaaggagttc
     4681 gaggactcga tcctgttctc gtaccaggtg gactcgaagt tcaaccgcaa gatctcggac
     4741 gccaccatct acgccacccg ccaggccaag gtgggcaagg acaaggccga cgagacctac
     4801 gtgctgggca agatcaagga catctacacc caggacggct acgacgcctt catgaagatc
     4861 tacaagaagg acaagtcgaa gttcctgatg taccgccacg acccgcagac cttcgagaag
     4921 gtgatcgagc cgatcctgga gaactacccg aacaagcaga tcaacgacaa gggcaaggag
     4981 gtgccgtgca acccgttcct gaagtacaag gaggagcacg gctacatccg caagtactcg
     5041 aagaagggca acggcccgga gatcaagtcg ctgaagtact acgactcgaa gctgggcaac
     5101 cacatcgaca tcaccccgaa ggactcgaac aacaaggtgg tgctgcagtc ggtgtcgccg
     5161 tggcgcgccg acgtgtactt caacaagacc accggcaagt acgagatcct gggcctgaag
     5221 tacgccgacc tgcagttcga caagggcacc ggcacctaca agatctcgca ggagaagtac
     5281 aacgacatca agaagaagga gggcgtggac tcggactcgg agttcaagtt caccctgtac
     5341 aagaacgacc tgctgctggt gaaggacacc gagaccaagg agcagcagct gttccgcttc
     5401 ctgtcgcgca ccatgccgaa gcagaagcac tacgtggagc tgaagccgta cgacaagcag
     5461 aagttcgagg gcggcgaggc cctgatcaag gtgctgggca acgtggccaa ctcgggccag
     5521 tgcaagaagg gcctgggcaa gtcgaacatc tcgatctaca aggtgcgcac cgacgtgctg
     5581 ggcaaccagc acatcatcaa gaacgagggc gacaagccga agctggactt ctaaatactc
     5641 tggccatttt tctttaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     5701 gagcgcaacg cgtgcggccg ctgattagct aagcagaagg ccatcctgac ggatggcctt
     5761 tttctagaaa aaaaaaaagc gccgcaactg cggcgctttt ttttttttaa ttaagtgata
     5821 gattgcgatg cgtgtgctcg gttgatataa tctgtcagat caggaggact tcgcatgtcg
     5881 cggttggata agtccaaggt catcaactcg gcgctggagc tgctgaacga ggtcggtatc
     5941 gagggactga ccacgcgcaa gttggcccaa aagctcggcg tggaacaacc gaccctctac
     6001 tggcacgtga agaacaagcg cgcgctgctg gatgcgttgg ccatcgagat gctggatcgc
     6061 catcacacgc acttttgccc cctggagggc gagagctggc aggattttct gcggaacaac
     6121 gcgaagtcgt tccggtgcgc cctcctgagc catcgggatg gcgccaaagt ccacttgggc
     6181 acccgcccga ccgagaagca atacgagacc ctggagaacc agctggcctt cctctgtcaa
     6241 cagggttttt cgttggagaa cgccctgtat gccctgtccg ccgtgggaca cttcaccttg
     6301 ggctgcgtgt tggaggatca ggaacatcag gtggcgaaag aggaacggga gacccccacg
     6361 acggattcga tgcccccgtt gttgcgccag gcgatcgaac tgtttgacca ccagggcgcc
     6421 gaacccgcct ttttgttcgg gctggagctg atcatctgcg ggctggaaaa gcaactgaag
     6481 tgcgaaagcg gttcgtgata acgccgaaaa ccccgcttcg gcggggtttt gccgcggtct
     6541 gcgttgtcgg gaagatttaa aataaaaaag gggacctcta gggtccccaa ttaaatagtt
     6601 atagaattcg cgtttaatac tgtttaaact ctagaaatat tggatcgtcg gcaccgtcac
     6661 ggccgtggga ggcggcacga tccgcgacgt gatgatcggc cgcatcccca cggtgctgcg
     6721 cagtgagctc tacgccatcc cggcgttgat ctgtgcgttc gcacgcacag gcccggtgtg
     6781 agaagggtct ctgacgagcg ggagaaccca cccggggtgg gcgagtttgt cctgcgtgtg
     6841 ctcggtcgag taggctctgg gagtacccgt gtgtacgacc agcacggcat acatcatttc
     6901 gacgccgaga gattcgccgc ccgaaatgag cacgatccgc atgcttaatt aaggaggcac
     6961 cggtatggtg agcaagggcg aggaggtcat caaagagttc atgcgcttca aggtgcgcat
     7021 ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     7081 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
     7141 ggacatcctg tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga
     7201 catccccgat tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     7261 cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct
     7321 gatctacaag gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa
     7381 gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa
     7441 gggcgagatc caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa
     7501 gaccatctac atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa
     7561 gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga
     7621 gggccgccac cacctgttcc tgtacggcat ggacgagctg tacaagtaga agctttataa
     7681 acactagtat taaaaaaaaa agccccgcga ttgcggggct tttttttttc gacggcgagg
     7741 gttccgaccg ctgcaactcc cggtgcaacc ttgtcccggt ctattctctt cactgcacca
     7801 gctccaatct ggtgtgaatg cccctcgtct gttcgcgcag gcggggggct ctattcgttt
     7861 gtcagcatcg aaagtagcca gatcagggat gcgttgcaac cgcgtatgcc caggtcagaa
     7921 gagtcgcaca agagttgcag acccctggaa agaaaaatgg ccagagggcg aaaacaccct
     7981 ctgaccagcg gagcgggcga cgggaatcga acccgcgtag ctagtttgga agaatgggtg
     8041 tctgccgacc acatatatgg gccggtcaag ataggttttt accccctctc ggctgcatcc
     8101 tctaagtgga aagaaattgc aggtcgtaga agcgcgttga agcctgagag ttgcacagga
     8161 gttgcaaccc ggtagccttg ttcacgacga gaggagacct agttggcacg tcgcggatgg
     8221 ggatcgctga agactcagcg cagcgggagg atccaagcct catacgtcaa cccgcaggac
     8281 ggtgtgaggt actacgcgct gcagacctac gacaacaaga tggacgccga agcctggctc
     8341 gcgggcgaga agcggctcat cgagatggag acctggaccc ctccacagga ccgggcgaag
     8401 aaggcagccg ccagcgccat cacgctggag gagtacaccc ggaagtggct cgtggagcgc
     8461 gacctcgcag acggcaccag ggatctgtac agcgggcacg cggagcgccg catctacccg
     8521 gtgctaggtg aagtggcggt cacagagatg acgccagctc tggtgcgtgc gtggtgggcc
     8581 gggatgggta ggaagcaccc gactgcccgc cggcatgcct acaacgtcct ccgggcggtg
     8641 atgaacacag cggtcgagga caagctgatc gcagagaacc cgtgccggat cgagcagaag
     8701 gcagccgatg agcgcgacgt agaggcgctg acgcctgagg agctggacat cgtcgccgct
     8761 gagatcttcg tgcactaccg gatcgcggca tacatcctgg cgtggacgag cctccggttc
     8821 ggagagctga tcgagcttcg ccgcaaggac atcgtggacg acggcatgac gatgaagctc
     8881 cgggtgcgcc gtggcgcttc ccgcgtgggg aacaagatcg tcgttggcaa cgccaagacc
     8941 gtccggtcga agcgtcctgt gacggttccg cctcacgtcg cggagatgat ccgagcgcac
     9001 atgaaggacc gtacgaagat gaacaagggc cccgaggcat tcctggtgac cacgacgcag
     9061 ggcaaccggc tgtcgaagtc cgcgttcacc aagtcgctga agcgtggcta cgccaagatc
     9121 ggtcggccgg aactccgcat ccacgacctc cgcgctgtcg gcgctacgtt cgccgctcag
     9181 gcaggtgcga cgaccaagga gctgatggcc cgcctcggtc acacgactcc taggatggcg
     9241 atgaagtacc agatggcgtc tgaggcccgc gacgaggcta tcgctgaggc gatgtccaag
     9301 ctggccaaga cctcctgaaa cgcaaaaagc ccccctccca aggacactga gtcctaaaga
     9361 ggggggtttc ttgtcagtac gcgaagaacc acgcctggcc gcgagcgcca gcaccgccgc
     9421 tctgtgcgga gacctgggca ccagccccgc cgccgccagg agcattgccg ttcccgccag
     9481 ctagccacct gacgtcgggg gggggggaaa gccacgttgt gtctcaaaat ctctgatgtt
     9541 acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca
     9601 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctcg aggccgcgat
     9661 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
//
