LOCUS       sequence-455967-        9717 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kanamycin resistant L5-integrative vector for CRISPR interference in
            M.smegmatis with constitutive expression of mWasabi under the 
            control of the psmyc promotor (psmyc::mWasabi).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9717)
  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 9717)
  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: pJL31
FEATURES             Location/Qualifiers
     source          1..9717
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2176..2203
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3052..3762)
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYK"
     primer_bind     complement(6055..6074)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6066..6881
                     /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(6139..6158)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(7594..8182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7674..7693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    8365..8383
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8395..8413
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 tgatgatgga gctgatcccg gagctgtacg agacctcgga ggagcagatg accatcctga
       61 cccgcctggg caagcagaag accacctcgt cgtcgaacaa gaccaagtac atcgacgaga
      121 agctgctgac cgaggagatc tacaacccgg tggtggccaa gtcggtgcgc caggccatca
      181 agatcgtgaa cgccgccatc aaggagtacg gcgacttcga caacatcgtg atcgagatgg
      241 cccgcgagac caacgaggac gacgagaaga aggccatcca gaagatccag aaggccaaca
      301 aggacgagaa ggacgccgcc atgctgaagg ccgccaacca gtacaacggc aaggccgagc
      361 tgccgcactc ggtgttccac ggccacaagc agctggccac caagatccgc ctgtggcacc
      421 agcagggcga gcgctgcctg tacaccggca agaccatctc gatccacgac ctgatcaaca
      481 actcgaacca gttcgaggtg gacgccatcc tgccgctgtc gatcaccttc gacgactcgc
      541 tggccaacaa ggtgctggtg tacgccaccg ccaaccagga gaagggccag cgcaccccgt
      601 accaggccct ggactcgatg gacgacgcct ggtcgttccg cgagctgaag gccttcgtgc
      661 gcgagtcgaa gaccctgtcg aacaagaaga aggagtacct gctgaccgag gaggacatct
      721 cgaagttcga cgtgcgcaag aagttcatcg agcgcaacct ggtggacacc cgctacgcct
      781 cgcgcgtggt gctgaacgcc ctgcaggagc acttccgcgc ccacaagatc gacaccaagg
      841 tgtcggtggt gcgcggccag ttcacctcgc agctgcgccg ccactggggc atcgagaaga
      901 cccgcgacac ctaccaccac cacgccgtgg acgccctgat catcgccgcc tcgtcgcagc
      961 tgaacctgtg gaagaagcag aagaacaccc tggtgtcgta ctcggaggac cagctgctgg
     1021 acatcgagac cggcgagctg atctcggacg acgagtacaa ggagtcggtg ttcaaggccc
     1081 cgtaccagca cttcgtggac accctgaagt cgaaggagtt cgaggactcg atcctgttct
     1141 cgtaccaggt ggactcgaag ttcaaccgca agatctcgga cgccaccatc tacgccaccc
     1201 gccaggccaa ggtgggcaag gacaaggccg acgagaccta cgtgctgggc aagatcaagg
     1261 acatctacac ccaggacggc tacgacgcct tcatgaagat ctacaagaag gacaagtcga
     1321 agttcctgat gtaccgccac gacccgcaga ccttcgagaa ggtgatcgag ccgatcctgg
     1381 agaactaccc gaacaagcag atcaacgaca agggcaagga ggtgccgtgc aacccgttcc
     1441 tgaagtacaa ggaggagcac ggctacatcc gcaagtactc gaagaagggc aacggcccgg
     1501 agatcaagtc gctgaagtac tacgactcga agctgggcaa ccacatcgac atcaccccga
     1561 aggactcgaa caacaaggtg gtgctgcagt cggtgtcgcc gtggcgcgcc gacgtgtact
     1621 tcaacaagac caccggcaag tacgagatcc tgggcctgaa gtacgccgac ctgcagttcg
     1681 acaagggcac cggcacctac aagatctcgc aggagaagta caacgacatc aagaagaagg
     1741 agggcgtgga ctcggactcg gagttcaagt tcaccctgta caagaacgac ctgctgctgg
     1801 tgaaggacac cgagaccaag gagcagcagc tgttccgctt cctgtcgcgc accatgccga
     1861 agcagaagca ctacgtggag ctgaagccgt acgacaagca gaagttcgag ggcggcgagg
     1921 ccctgatcaa ggtgctgggc aacgtggcca actcgggcca gtgcaagaag ggcctgggca
     1981 agtcgaacat ctcgatctac aaggtgcgca ccgacgtgct gggcaaccag cacatcatca
     2041 agaacgaggg cgacaagccg aagctggact tctaaatact ctggccattt ttctttaccg
     2101 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcaac gcgtgcggcc
     2161 gctgattagc taagcagaag gccatcctga cggatggcct ttttctagaa aaaaaaaaag
     2221 cgccgcaact gcggcgcttt ttttttttta attaagtgat agattgcgat gcgtgtgctc
     2281 ggttgatata atctgtcaga tcaggaggac ttcgcatgtc gcggttggat aagtccaagg
     2341 tcatcaactc ggcgctggag ctgctgaacg aggtcggtat cgagggactg accacgcgca
     2401 agttggccca aaagctcggc gtggaacaac cgaccctcta ctggcacgtg aagaacaagc
     2461 gcgcgctgct ggatgcgttg gccatcgaga tgctggatcg ccatcacacg cacttttgcc
     2521 ccctggaggg cgagagctgg caggattttc tgcggaacaa cgcgaagtcg ttccggtgcg
     2581 ccctcctgag ccatcgggat ggcgccaaag tccacttggg cacccgcccg accgagaagc
     2641 aatacgagac cctggagaac cagctggcct tcctctgtca acagggtttt tcgttggaga
     2701 acgccctgta tgccctgtcc gccgtgggac acttcacctt gggctgcgtg ttggaggatc
     2761 aggaacatca ggtggcgaaa gaggaacggg agacccccac gacggattcg atgcccccgt
     2821 tgttgcgcca ggcgatcgaa ctgtttgacc accagggcgc cgaacccgcc tttttgttcg
     2881 ggctggagct gatcatctgc gggctggaaa agcaactgaa gtgcgaaagc ggttcgtgat
     2941 aacgccgaaa accccgcttc ggcggggttt tgccgcggtc tgcgttgtcg ggaagattta
     3001 aaataaaaaa ggggacctct agggtcccca attaaatagt ttataaagct tttacttgta
     3061 cagctcgtcc atgccgtcgg tggagttgcg ggccacggcg atctcgtaaa cggtcacctt
     3121 gttgtagtcc ttgtcgtggt tcaggatctc gatgcggtgg tccacaaagt gatagtcggg
     3181 cagcttcacc gccttcttgg ccctgtagat ggtcttgaag tcaacgcggt ggtggccgcc
     3241 gccctccagc agcagcttca tcttgacgtc gcccttcagc acgccgtcgc gcacgtacat
     3301 cctctcggtg gaggcgtccc agccggtggt ctccttctgc atcacggggc cgttgggggg
     3361 gaagttctcg cccttgaggt gtatctcgta gatgaaggag tcctcctcca tggagatgtc
     3421 ggacttcacc ttcacgatgc ccttgtcctc gaaggtcatg gtgcgctccc aagagtagcc
     3481 ctcggggaag gactgcttga agtagttggg gatgtcgtcg gggtacttgg tgaaggccct
     3541 gttgccgtaa ctgaacgcgg tggtcagaat gtcgtaggag aagggcaggg gggctccctc
     3601 cttcacctcc aggttgatgg tgttggtgcc gtcgtagggc ttgccctcgc cctcgccctc
     3661 gatcacgaag gcgtggccat tcacgttgcc ctccatcttc agcttgatct tcatgtcggg
     3721 cttgattacg cccattgtgg tctcctcgcc cttgctcacc ataccggtgc ctccttaatt
     3781 aagcatgcgg atcgtgctca tttcgggcgg cgaatctctc ggcgtcgaaa tgatgtatgc
     3841 cgtgctggtc gtacacacgg gtactcccag agcctactcg accgagcaca cgcaggacaa
     3901 actcgcccac cccgggtggg ttctcccgct cgtcagagac ccttctcaca ccgggcctgt
     3961 gcgtgcgaac gcacagatca acgccgggat ggcgtagagc tcactgcgca gcaccgtggg
     4021 gatgcggccg atcatcacgt cgcggatcgt gccgcctccc acggccgtga cggtgccgac
     4081 gatccaatat ttctagagtt taaacagtat taaacgcgaa ttctataaac actagtatta
     4141 aaaaaaaaag ccccgcgatt gcggggcttt tttttttcga cggcgagggt tccgaccgct
     4201 gcaactcccg gtgcaacctt gtcccggtct attctcttca ctgcaccagc tccaatctgg
     4261 tgtgaatgcc cctcgtctgt tcgcgcaggc ggggggctct attcgtttgt cagcatcgaa
     4321 agtagccaga tcagggatgc gttgcaaccg cgtatgccca ggtcagaaga gtcgcacaag
     4381 agttgcagac ccctggaaag aaaaatggcc agagggcgaa aacaccctct gaccagcgga
     4441 gcgggcgacg ggaatcgaac ccgcgtagct agtttggaag aatgggtgtc tgccgaccac
     4501 atatatgggc cggtcaagat aggtttttac cccctctcgg ctgcatcctc taagtggaaa
     4561 gaaattgcag gtcgtagaag cgcgttgaag cctgagagtt gcacaggagt tgcaacccgg
     4621 tagccttgtt cacgacgaga ggagacctag ttggcacgtc gcggatgggg atcgctgaag
     4681 actcagcgca gcgggaggat ccaagcctca tacgtcaacc cgcaggacgg tgtgaggtac
     4741 tacgcgctgc agacctacga caacaagatg gacgccgaag cctggctcgc gggcgagaag
     4801 cggctcatcg agatggagac ctggacccct ccacaggacc gggcgaagaa ggcagccgcc
     4861 agcgccatca cgctggagga gtacacccgg aagtggctcg tggagcgcga cctcgcagac
     4921 ggcaccaggg atctgtacag cgggcacgcg gagcgccgca tctacccggt gctaggtgaa
     4981 gtggcggtca cagagatgac gccagctctg gtgcgtgcgt ggtgggccgg gatgggtagg
     5041 aagcacccga ctgcccgccg gcatgcctac aacgtcctcc gggcggtgat gaacacagcg
     5101 gtcgaggaca agctgatcgc agagaacccg tgccggatcg agcagaaggc agccgatgag
     5161 cgcgacgtag aggcgctgac gcctgaggag ctggacatcg tcgccgctga gatcttcgtg
     5221 cactaccgga tcgcggcata catcctggcg tggacgagcc tccggttcgg agagctgatc
     5281 gagcttcgcc gcaaggacat cgtggacgac ggcatgacga tgaagctccg ggtgcgccgt
     5341 ggcgcttccc gcgtggggaa caagatcgtc gttggcaacg ccaagaccgt ccggtcgaag
     5401 cgtcctgtga cggttccgcc tcacgtcgcg gagatgatcc gagcgcacat gaaggaccgt
     5461 acgaagatga acaagggccc cgaggcattc ctggtgacca cgacgcaggg caaccggctg
     5521 tcgaagtccg cgttcaccaa gtcgctgaag cgtggctacg ccaagatcgg tcggccggaa
     5581 ctccgcatcc acgacctccg cgctgtcggc gctacgttcg ccgctcaggc aggtgcgacg
     5641 accaaggagc tgatggcccg cctcggtcac acgactccta ggatggcgat gaagtaccag
     5701 atggcgtctg aggcccgcga cgaggctatc gctgaggcga tgtccaagct ggccaagacc
     5761 tcctgaaacg caaaaagccc ccctcccaag gacactgagt cctaaagagg ggggtttctt
     5821 gtcagtacgc gaagaaccac gcctggccgc gagcgccagc accgccgctc tgtgcggaga
     5881 cctgggcacc agccccgccg ccgccaggag cattgccgtt cccgccagct agccacctga
     5941 cgtcgggggg gggggaaagc cacgttgtgt ctcaaaatct ctgatgttac attgcacaag
     6001 ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     6061 gtgttatgag ccatattcaa cgggaaacgt cttgctcgag gccgcgatta aattccaaca
     6121 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     6181 caatctatcg cttgtatggg aagccccatg cgccagagtt gtttctgaaa catggcaaag
     6241 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     6301 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     6361 ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     6421 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     6481 gtccttttaa cagcgatcgc gtatttcgcc tcgctcaggc gcaatcacga atgaataacg
     6541 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     6601 ggaaagaaat gcataatctt ttgccattct caccggattc agtcgtcact catggtgatt
     6661 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     6721 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     6781 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     6841 ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt
     6901 tgtaacactg gcagagcatt acgctgactt gacgggacgg cggctttgtt gaataaatcg
     6961 aacttttgct gagttgaagg atcagatcac gcatcttccc gacaacgcag accgttccgt
     7021 ggcaaagcaa aagttcaaaa tcaccaactg gtccacctac aacaaagctc tcaccaaccg
     7081 tggctccctc acgatatcaa taaacgaaag gctcagtcga aagactgggc ctttcgttta
     7141 tctgttgttt gaaaaaaaaa agcgccgcaa ctgcggcgct tttttttttg aattctctga
     7201 ccagggaaaa tagccctctg acctggggat ttgcgatccc tatcagtgat agatataatc
     7261 tgggaggaga cgattaatgc gtctcggttt ttgtactcga aagaagctac aaagataagg
     7321 cttcatgccg aaatcaacac cctgtcattt tatggcaggg tgtttttttt ttgtcgactt
     7381 ggggacccta gaggtcccct tttttatttt tttggatgaa gagcattatc aatggctctt
     7441 cacaggaagc ttaaaaaaaa aagccccgcg attgcggggc tttttttttt taatacggtt
     7501 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     7561 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     7621 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     7681 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7741 cggatacctg tccgcctttc tcccctcggg aagcgtggcg ctttctcata gctcacgctg
     7801 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7861 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7921 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7981 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     8041 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     8101 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     8161 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     8221 tggaacaata aacgaaaggc tcagtcgaaa gactgggcct ttcgtttatc tgttgtttga
     8281 aaaaaaaaag ccccgcgatt gcggggcttt tttttttcat atgtctgacc agggaaaata
     8341 gccctctgac ctggggattt gacttcccta tcagtgatag agataatctg ggagtcccta
     8401 tcagtgatag agaaggcgct atcgatatgt cggacctggt gctgggcctg gccatcggca
     8461 tcggctcggt gggcgtgggc atcctgaaca aggtgaccgg cgagatcatc cacaagaact
     8521 cgcgcatctt cccggccgcc caggccgaga acaacctggt gcgccgcacc aaccgccagg
     8581 gccgccgcct ggcccgccgc aagaagcacc gccgcgtgcg cctgaaccgc ctgttcgagg
     8641 agtcgggcct gatcaccgac ttcaccaaga tctcgatcaa cctgaacccg taccagctgc
     8701 gcgtgaaggg cctgaccgac gagctgtcga acgaggagct gttcatcgcc ctgaagaaca
     8761 tggtgaagca ccgcggcatc tcgtacctgg acgacgcctc ggacgacggc aactcgtcgg
     8821 tgggcgacta cgcccagatc gtgaaggaga actcgaagca gctggagacc aagaccccgg
     8881 gccagatcca gctggagcgc taccagacct acggccagct gcgcggcgac ttcaccgtgg
     8941 agaaggacgg caagaagcac cgcctgatca acgtgttccc gacctcggcc taccgctcgg
     9001 aggccctgcg catcctgcag acccagcagg agttcaaccc gcagatcacc gacgagttca
     9061 tcaaccgcta cctggagatc ctgaccggca agcgcaagta ctaccacggc ccgggcaacg
     9121 agaagtcgcg caccgactac ggccgctacc gcacctcggg cgagaccctg gacaacatct
     9181 tcggcatcct gatcggcaag tgcaccttct acccggacga gttccgcgcc gccaaggcct
     9241 cgtacaccgc ccaggagttc aacctgctga acgacctgaa caacctgacc gtgccgaccg
     9301 agaccaagaa gctgtcgaag gagcagaaga accagatcat caactacgtg aagaacgaga
     9361 aggccatggg cccggccaag ctgttcaagt acatcgccaa gctgctgtcg tgcgacgtgg
     9421 ccgacatcaa gggctaccgc atcgacaagt cgggcaaggc cgagatccac accttcgagg
     9481 cctaccgcaa gatgaagacc ctggagaccc tggacatcga gcagatggac cgcgagaccc
     9541 tggacaagct ggcctacgtg ctgaccctga acaccgagcg cgagggcatc caggaggccc
     9601 tggagcacga gttcgccgac ggctcgttct cgcagaagca ggtggacgag ctggtgcagt
     9661 tccgcaaggc caactcgtcg atcttcggca agggctggca caacttctcg gtgaagc
//
