LOCUS       sequence-455969-        9716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kanamycin resistant L5-integrative vector for CRISPR interference in
            M.tuberculosis with the 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 9716)
  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 9716)
  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: pJL33
FEATURES             Location/Qualifiers
     source          1..9716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    218..236
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    248..266
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      449..1037
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     938..957
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1750..2565)
                     /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     2473..2492
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2557..2576
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4868..5578
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYK"
     terminator      complement(6427..6454)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcggttcagg cgcacgcggc ggtgcttctt gcggcgggcc aggcggcggc cctggcggtt
       61 ggtgcggcgc accaggttgt tctcggcctg ggcggccggg aagatgcgcg agttcttgtg
      121 gatgatctcg ccggtcacct tgttcaggat gcccacgccc accgagccga tgccgatggc
      181 caggcccagc accaggtccg acatatcgat accgcgttct ctatcactga tagggactcc
      241 cagattatct ctatcactga tagggaagtc aaatccccag gtcagagggc tattttccct
      301 ggtcagacat atgaaaaaaa aaagccccgc aatcgcgggg cttttttttt tcaaacaaca
      361 gataaacgaa aggcccagtc tttcgactga gcctttcgtt tattgttcca tgagcgtcag
      421 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
      481 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
      541 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
      601 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
      661 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
      721 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
      781 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
      841 tatgagaaag cgccacgctt cccgagggga gaaaggcgga caggtatccg gtaagcggca
      901 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
      961 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     1021 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     1081 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     1141 aaaaaaaaaa gccccgcaat cgcggggctt ttttttttaa gcttcctgtg aagagccatt
     1201 gataatgctc ttcatccaaa aaaataaaaa aggggacctc tagggtcccc aagtcgacaa
     1261 aaaaaaaaca ccctgccata aaatgacagg gtgttgattt cggcatgaag ccttatcttt
     1321 gtagcttctt tcgagtacaa aaaccgagac gcattaatcg tctcctccca gattatatct
     1381 atcactgata gggatcgcaa atccccaggt cagagggcta ttttccctgg tcagagaatt
     1441 caaaaaaaaa agcgccgcag ttgcggcgct tttttttttc aaacaacaga taaacgaaag
     1501 gcccagtctt tcgactgagc ctttcgttta ttgatatcgt gagggagcca cggttggtga
     1561 gagctttgtt gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt
     1621 ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc
     1681 aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac
     1741 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     1801 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     1861 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     1921 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     1981 agtgacgact gaatccggtg agaatggcaa aagattatgc atttctttcc agacttgttc
     2041 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     2101 tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac aattacaaac
     2161 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     2221 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     2281 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     2341 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     2401 tttcagaaac aactctggcg catggggctt cccatacaag cgatagattg tcgcacctga
     2461 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     2521 taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     2581 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     2641 gtaacatcag agattttgag acacaacgtg gctttccccc ccccccgacg tcaggtggct
     2701 agctggcggg aacggcaatg ctcctggcgg cggcggggct ggtgcccagg tctccgcaca
     2761 gagcggcggt gctggcgctc gcggccaggc gtggttcttc gcgtactgac aagaaacccc
     2821 cctctttagg actcagtgtc cttgggaggg gggctttttg cgtttcagga ggtcttggcc
     2881 agcttggaca tcgcctcagc gatagcctcg tcgcgggcct cagacgccat ctggtacttc
     2941 atcgccatcc taggagtcgt gtgaccgagg cgggccatca gctccttggt cgtcgcacct
     3001 gcctgagcgg cgaacgtagc gccgacagcg cggaggtcgt ggatgcggag ttccggccga
     3061 ccgatcttgg cgtagccacg cttcagcgac ttggtgaacg cggacttcga cagccggttg
     3121 ccctgcgtcg tggtcaccag gaatgcctcg gggcccttgt tcatcttcgt acggtccttc
     3181 atgtgcgctc ggatcatctc cgcgacgtga ggcggaaccg tcacaggacg cttcgaccgg
     3241 acggtcttgg cgttgccaac gacgatcttg ttccccacgc gggaagcgcc acggcgcacc
     3301 cggagcttca tcgtcatgcc gtcgtccacg atgtccttgc ggcgaagctc gatcagctct
     3361 ccgaaccgga ggctcgtcca cgccaggatg tatgccgcga tccggtagtg cacgaagatc
     3421 tcagcggcga cgatgtccag ctcctcaggc gtcagcgcct ctacgtcgcg ctcatcggct
     3481 gccttctgct cgatccggca cgggttctct gcgatcagct tgtcctcgac cgctgtgttc
     3541 atcaccgccc ggaggacgtt gtaggcatgc cggcgggcag tcgggtgctt cctacccatc
     3601 ccggcccacc acgcacgcac cagagctggc gtcatctctg tgaccgccac ttcacctagc
     3661 accgggtaga tgcggcgctc cgcgtgcccg ctgtacagat ccctggtgcc gtctgcgagg
     3721 tcgcgctcca cgagccactt ccgggtgtac tcctccagcg tgatggcgct ggcggctgcc
     3781 ttcttcgccc ggtcctgtgg aggggtccag gtctccatct cgatgagccg cttctcgccc
     3841 gcgagccagg cttcggcgtc catcttgttg tcgtaggtct gcagcgcgta gtacctcaca
     3901 ccgtcctgcg ggttgacgta tgaggcttgg atcctcccgc tgcgctgagt cttcagcgat
     3961 ccccatccgc gacgtgccaa ctaggtctcc tctcgtcgtg aacaaggcta ccgggttgca
     4021 actcctgtgc aactctcagg cttcaacgcg cttctacgac ctgcaatttc tttccactta
     4081 gaggatgcag ccgagagggg gtaaaaacct atcttgaccg gcccatatat gtggtcggca
     4141 gacacccatt cttccaaact agctacgcgg gttcgattcc cgtcgcccgc tccgctggtc
     4201 agagggtgtt ttcgccctct ggccattttt ctttccaggg gtctgcaact cttgtgcgac
     4261 tcttctgacc tgggcatacg cggttgcaac gcatccctga tctggctact ttcgatgctg
     4321 acaaacgaat agagcccccc gcctgcgcga acagacgagg ggcattcaca ccagattgga
     4381 gctggtgcag tgaagagaat agaccgggac aaggttgcac cgggagttgc agcggtcgga
     4441 accctcgccg tcgaaaaaaa aaagccccgc aatcgcgggg cttttttttt taatactagt
     4501 gttatagaat tcgcgtttaa tactgtttaa actctagaaa tattggatcg tcggcaccgt
     4561 cacggccgtg ggaggcggca cgatccgcga cgtgatgatc ggccgcatcc ccacggtgct
     4621 gcgcagtgag ctctacgcca tcccggcgtt gatctgtgcg ttcgcacgca caggcccggt
     4681 gtgagaaggg tctctgacga gcgggagaac ccacccgggg tgggcgagtt tgtcctgcgt
     4741 gtgctcggtc gagtaggctc tgggagtacc cgtgtgtacg accagcacgg catacatcat
     4801 ttcgacgccg agagattcgc cgcccgaaat gagcacgatc cgcatgctta attaaggagg
     4861 caccggtatg gtgagcaagg gcgaggagac cacaatgggc gtaatcaagc ccgacatgaa
     4921 gatcaagctg aagatggagg gcaacgtgaa tggccacgcc ttcgtgatcg agggcgaggg
     4981 cgagggcaag ccctacgacg gcaccaacac catcaacctg gaggtgaagg agggagcccc
     5041 cctgcccttc tcctacgaca ttctgaccac cgcgttcagt tacggcaaca gggccttcac
     5101 caagtacccc gacgacatcc ccaactactt caagcagtcc ttccccgagg gctactcttg
     5161 ggagcgcacc atgaccttcg aggacaaggg catcgtgaag gtgaagtccg acatctccat
     5221 ggaggaggac tccttcatct acgagataca cctcaagggc gagaacttcc cccccaacgg
     5281 ccccgtgatg cagaaggaga ccaccggctg ggacgcctcc accgagagga tgtacgtgcg
     5341 cgacggcgtg ctgaagggcg acgtcaagat gaagctgctg ctggagggcg gcggccacca
     5401 ccgcgttgac ttcaagacca tctacagggc caagaaggcg gtgaagctgc ccgactatca
     5461 ctttgtggac caccgcatcg agatcctgaa ccacgacaag gactacaaca aggtgaccgt
     5521 ttacgagatc gccgtggccc gcaactccac cgacggcatg gacgagctgt acaagtaaaa
     5581 gctttataaa ctatttaatt ggggacccta gaggtcccct tttttatttt aaatcttccc
     5641 gacaacgcag accgcggcaa aaccccgccg aagcggggtt ttcggcgtta tcacgaaccg
     5701 ctttcgcact tcagttgctt ttccagcccg cagatgatca gctccagccc gaacaaaaag
     5761 gcgggttcgg cgccctggtg gtcaaacagt tcgatcgcct ggcgcaacaa cgggggcatc
     5821 gaatccgtcg tgggggtctc ccgttcctct ttcgccacct gatgttcctg atcctccaac
     5881 acgcagccca aggtgaagtg tcccacggcg gacagggcat acagggcgtt ctccaacgaa
     5941 aaaccctgtt gacagaggaa ggccagctgg ttctccaggg tctcgtattg cttctcggtc
     6001 gggcgggtgc ccaagtggac tttggcgcca tcccgatggc tcaggagggc gcaccggaac
     6061 gacttcgcgt tgttccgcag aaaatcctgc cagctctcgc cctccagggg gcaaaagtgc
     6121 gtgtgatggc gatccagcat ctcgatggcc aacgcatcca gcagcgcgcg cttgttcttc
     6181 acgtgccagt agagggtcgg ttgttccacg ccgagctttt gggccaactt gcgcgtggtc
     6241 agtccctcga taccgacctc gttcagcagc tccagcgccg agttgatgac cttggactta
     6301 tccaaccgcg acatgcgaag tcctcctgat ctgacagatt atatcaaccg agcacacgca
     6361 tcgcaatcta tcacttaatt aaaaaaaaaa aagcgccgca gttgcggcgc tttttttttt
     6421 tctagaaaaa ggccatccgt caggatggcc ttctgcttag ctaatcagcg gccgcacgcg
     6481 ttgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaaagaaa
     6541 aatggccaga gtatttagaa gtccagcttc ggcttgtcgc cctcgttctt gatgatgtgc
     6601 tggttgccca gcacgtcggt gcgcaccttg tagatcgaga tgttcgactt gcccaggccc
     6661 ttcttgcact ggcccgagtt ggccacgttg cccagcacct tgatcagggc ctcgccgccc
     6721 tcgaacttct gcttgtcgta cggcttcagc tccacgtagt gcttctgctt cggcatggtg
     6781 cgcgacagga agcggaacag ctgctgctcc ttggtctcgg tgtccttcac cagcagcagg
     6841 tcgttcttgt acagggtgaa cttgaactcc gagtccgagt ccacgccctc cttcttcttg
     6901 atgtcgttgt acttctcctg cgagatcttg taggtgccgg tgcccttgtc gaactgcagg
     6961 tcggcgtact tcaggcccag gatctcgtac ttgccggtgg tcttgttgaa gtacacgtcg
     7021 gcgcgccacg gcgacaccga ctgcagcacc accttgttgt tcgagtcctt cggggtgatg
     7081 tcgatgtggt tgcccagctt cgagtcgtag tacttcagcg acttgatctc cgggccgttg
     7141 cccttcttcg agtacttgcg gatgtagccg tgctcctcct tgtacttcag gaacgggttg
     7201 cacggcacct ccttgccctt gtcgttgatc tgcttgttcg ggtagttctc caggatcggc
     7261 tcgatcacct tctcgaaggt ctgcgggtcg tggcggtaca tcaggaactt cgacttgtcc
     7321 ttcttgtaga tcttcatgaa ggcgtcgtag ccgtcctggg tgtagatgtc cttgatcttg
     7381 cccagcacgt aggtctcgtc ggccttgtcc ttgcccacct tggcctggcg ggtggcgtag
     7441 atggtggcgt ccgagatctt gcggttgaac ttcgagtcca cctggtacga gaacaggatc
     7501 gagtcctcga actccttcga cttcagggtg tccacgaagt gctggtacgg ggccttgaac
     7561 accgactcct tgtactcgtc gtccgagatc agctcgccgg tctcgatgtc cagcagctgg
     7621 tcctccgagt acgacaccag ggtgttcttc tgcttcttcc acaggttcag ctgcgacgag
     7681 gcggcgatga tcagggcgtc cacggcgtgg tggtggtagg tgtcgcgggt cttctcgatg
     7741 ccccagtggc ggcgcagctg cgaggtgaac tggccgcgca ccaccgacac cttggtgtcg
     7801 atcttgtggg cgcggaagtg ctcctgcagg gcgttcagca ccacgcgcga ggcgtagcgg
     7861 gtgtccacca ggttgcgctc gatgaacttc ttgcgcacgt cgaacttcga gatgtcctcc
     7921 tcggtcagca ggtactcctt cttcttgttc gacagggtct tcgactcgcg cacgaaggcc
     7981 ttcagctcgc ggaacgacca ggcgtcgtcc atcgagtcca gggcctggta cggggtgcgc
     8041 tggcccttct cctggttggc ggtggcgtac accagcacct tgttggccag cgagtcgtcg
     8101 aaggtgatcg acagcggcag gatggcgtcc acctcgaact ggttcgagtt gttgatcagg
     8161 tcgtggatcg agatggtctt gccggtgtac aggcagcgct cgccctgctg gtgccacagg
     8221 cggatcttgg tggccagctg cttgtggccg tggaacaccg agtgcggcag ctcggccttg
     8281 ccgttgtact ggttggcggc cttcagcatg gcggcgtcct tctcgtcctt gttggccttc
     8341 tggatcttct ggatggcctt cttctcgtcg tcctcgttgg tctcgcgggc catctcgatc
     8401 acgatgttgt cgaagtcgcc gtactccttg atggcggcgt tcacgatctt gatggcctgg
     8461 cgcaccgact tggccaccac cgggttgtag atctcctcgg tcagcagctt ctcgtcgatg
     8521 tacttggtct tgttcgacga cgaggtggtc ttctgcttgc ccaggcgggt caggatggtc
     8581 atctgctcct ccgaggtctc gtacagctcc gggatcagct ccatcatcag cttcaccgag
     8641 aagttgtgcc agcccttgcc gaagatcgac gagttggcct tgcggaactg caccagctcg
     8701 tccacctgct tctgcgagaa cgagccgtcg gcgaactcgt gctccagggc ctcctggatg
     8761 ccctcgcgct cggtgttcag ggtcagcacg taggccagct tgtccagggt ctcgcggtcc
     8821 atctgctcga tgtccagggt ctccagggtc ttcatcttgc ggtaggcctc gaaggtgtgg
     8881 atctcggcct tgcccgactt gtcgatgcgg tagcccttga tgtcggccac gtcgcacgac
     8941 agcagcttgg cgatgtactt gaacagcttg gccgggccca tggccttctc gttcttcacg
     9001 tagttgatga tctggttctt ctgctccttc gacagcttct tggtctcggt cggcacggtc
     9061 aggttgttca ggtcgttcag caggttgaac tcctgggcgg tgtacgaggc cttggcggcg
     9121 cggaactcgt ccgggtagaa ggtgcacttg ccgatcagga tgccgaagat gttgtccagg
     9181 gtctcgcccg aggtgcggta gcggccgtag tcggtgcgcg acttctcgtt gcccgggccg
     9241 tggtagtact tgcgcttgcc ggtcaggatc tccaggtagc ggttgatgaa ctcgtcggtg
     9301 atctgcgggt tgaactcctg ctgggtctgc aggatgcgca gggcctccga gcggtaggcc
     9361 gaggtcggga acacgttgat caggcggtgc ttcttgccgt ccttctccac ggtgaagtcg
     9421 ccgcgcagct ggccgtaggt ctggtagcgc tccagctgga tctggcccgg ggtcttggtc
     9481 tccagctgct tcgagttctc cttcacgatc tgggcgtagt cgcccaccga cgagttgccg
     9541 tcgtccgagg cgtcgtccag gtacgagatg ccgcggtgct tcaccatgtt cttcagggcg
     9601 atgaacagct cctcgttcga cagctcgtcg gtcaggccct tcacgcgcag ctggtacggg
     9661 ttcaggttga tcgagatctt ggtgaagtcg gtgatcaggc ccgactcctc gaacag
//
