LOCUS       sequence-483390-        8633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi gene silencing plasmid with sgRNA (Rv1633) dCas9 TetR and 
            KanR L5 Int attP for M. tuberculosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8633)
  TITLE     Rhee Lab CRISPRi plasmids
REFERENCE   2  (bases 1 to 8633)
  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"
COMMENT     Sequence Label: PLJR965_Rv1633
FEATURES             Location/Qualifiers
     source          1..8633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(498..1086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(578..597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1269..1287
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1299..1317
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      4797..4824
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(7590..7609)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7601..8416
                     /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(7674..7693)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 aataaacgaa aggctcagtc gaaagactgg gcctttcgtt tatctgttgt ttgaaaaaaa
       61 aaagcgccgc aactgcggcg cttttttttt tgaattctct gaccagggaa aatagccctc
      121 tgacctgggg atttgcgatc cctatcagtg atagatataa tctgggaaca tgtgactcgt
      181 cgataacgag gtttttgtac tcgaaagaag ctacaaagat aaggcttcat gccgaaatca
      241 acaccctgtc attttatggc agggtgtttt ttttttgtcg acttggggac cctagaggtc
      301 ccctttttta tttttttgga tgaagagcat tatcaatggc tcttcacagg aagcttaaaa
      361 aaaaaagccc cgcgattgcg gggctttttt tttttaatac ggttatccac agaatcaggg
      421 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
      481 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
      541 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
      601 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
      661 tttctcccct cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
      721 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
      781 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
      841 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
      901 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
      961 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     1021 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1081 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcatggaac aataaacgaa
     1141 aggctcagtc gaaagactgg gcctttcgtt tatctgttgt ttgaaaaaaa aaagccccgc
     1201 gattgcgggg cttttttttt tcatatgtct gaccagggaa aatagccctc tgacctgggg
     1261 atttgacttc cctatcagtg atagagataa tctgggagtc cctatcagtg atagagaacg
     1321 cggtatcgat atgtcggacc tggtgctggg cctggccatc ggcatcggct cggtgggcgt
     1381 gggcatcctg aacaaggtga ccggcgagat catccacaag aactcgcgca tcttcccggc
     1441 cgcccaggcc gagaacaacc tggtgcgccg caccaaccgc cagggccgcc gcctggcccg
     1501 ccgcaagaag caccgccgcg tgcgcctgaa ccgcctgttc gaggagtcgg gcctgatcac
     1561 cgacttcacc aagatctcga tcaacctgaa cccgtaccag ctgcgcgtga agggcctgac
     1621 cgacgagctg tcgaacgagg agctgttcat cgccctgaag aacatggtga agcaccgcgg
     1681 catctcgtac ctggacgacg cctcggacga cggcaactcg tcggtgggcg actacgccca
     1741 gatcgtgaag gagaactcga agcagctgga gaccaagacc ccgggccaga tccagctgga
     1801 gcgctaccag acctacggcc agctgcgcgg cgacttcacc gtggagaagg acggcaagaa
     1861 gcaccgcctg atcaacgtgt tcccgacctc ggcctaccgc tcggaggccc tgcgcatcct
     1921 gcagacccag caggagttca acccgcagat caccgacgag ttcatcaacc gctacctgga
     1981 gatcctgacc ggcaagcgca agtactacca cggcccgggc aacgagaagt cgcgcaccga
     2041 ctacggccgc taccgcacct cgggcgagac cctggacaac atcttcggca tcctgatcgg
     2101 caagtgcacc ttctacccgg acgagttccg cgccgccaag gcctcgtaca ccgcccagga
     2161 gttcaacctg ctgaacgacc tgaacaacct gaccgtgccg accgagacca agaagctgtc
     2221 gaaggagcag aagaaccaga tcatcaacta cgtgaagaac gagaaggcca tgggcccggc
     2281 caagctgttc aagtacatcg ccaagctgct gtcgtgcgac gtggccgaca tcaagggcta
     2341 ccgcatcgac aagtcgggca aggccgagat ccacaccttc gaggcctacc gcaagatgaa
     2401 gaccctggag accctggaca tcgagcagat ggaccgcgag accctggaca agctggccta
     2461 cgtgctgacc ctgaacaccg agcgcgaggg catccaggag gccctggagc acgagttcgc
     2521 cgacggctcg ttctcgcaga agcaggtgga cgagctggtg cagttccgca aggccaactc
     2581 gtcgatcttc ggcaagggct ggcacaactt ctcggtgaag ctgatgatgg agctgatccc
     2641 ggagctgtac gagacctcgg aggagcagat gaccatcctg acccgcctgg gcaagcagaa
     2701 gaccacctcg tcgtcgaaca agaccaagta catcgacgag aagctgctga ccgaggagat
     2761 ctacaacccg gtggtggcca agtcggtgcg ccaggccatc aagatcgtga acgccgccat
     2821 caaggagtac ggcgacttcg acaacatcgt gatcgagatg gcccgcgaga ccaacgagga
     2881 cgacgagaag aaggccatcc agaagatcca gaaggccaac aaggacgaga aggacgccgc
     2941 catgctgaag gccgccaacc agtacaacgg caaggccgag ctgccgcact cggtgttcca
     3001 cggccacaag cagctggcca ccaagatccg cctgtggcac cagcagggcg agcgctgcct
     3061 gtacaccggc aagaccatct cgatccacga cctgatcaac aactcgaacc agttcgaggt
     3121 ggacgccatc ctgccgctgt cgatcacctt cgacgactcg ctggccaaca aggtgctggt
     3181 gtacgccacc gccaaccagg agaagggcca gcgcaccccg taccaggccc tggactcgat
     3241 ggacgacgcc tggtcgttcc gcgagctgaa ggccttcgtg cgcgagtcga agaccctgtc
     3301 gaacaagaag aaggagtacc tgctgaccga ggaggacatc tcgaagttcg acgtgcgcaa
     3361 gaagttcatc gagcgcaacc tggtggacac ccgctacgcc tcgcgcgtgg tgctgaacgc
     3421 cctgcaggag cacttccgcg cccacaagat cgacaccaag gtgtcggtgg tgcgcggcca
     3481 gttcacctcg cagctgcgcc gccactgggg catcgagaag acccgcgaca cctaccacca
     3541 ccacgccgtg gacgccctga tcatcgccgc ctcgtcgcag ctgaacctgt ggaagaagca
     3601 gaagaacacc ctggtgtcgt actcggagga ccagctgctg gacatcgaga ccggcgagct
     3661 gatctcggac gacgagtaca aggagtcggt gttcaaggcc ccgtaccagc acttcgtgga
     3721 caccctgaag tcgaaggagt tcgaggactc gatcctgttc tcgtaccagg tggactcgaa
     3781 gttcaaccgc aagatctcgg acgccaccat ctacgccacc cgccaggcca aggtgggcaa
     3841 ggacaaggcc gacgagacct acgtgctggg caagatcaag gacatctaca cccaggacgg
     3901 ctacgacgcc ttcatgaaga tctacaagaa ggacaagtcg aagttcctga tgtaccgcca
     3961 cgacccgcag accttcgaga aggtgatcga gccgatcctg gagaactacc cgaacaagca
     4021 gatcaacgac aagggcaagg aggtgccgtg caacccgttc ctgaagtaca aggaggagca
     4081 cggctacatc cgcaagtact cgaagaaggg caacggcccg gagatcaagt cgctgaagta
     4141 ctacgactcg aagctgggca accacatcga catcaccccg aaggactcga acaacaaggt
     4201 ggtgctgcag tcggtgtcgc cgtggcgcgc cgacgtgtac ttcaacaaga ccaccggcaa
     4261 gtacgagatc ctgggcctga agtacgccga cctgcagttc gacaagggca ccggcaccta
     4321 caagatctcg caggagaagt acaacgacat caagaagaag gagggcgtgg actcggactc
     4381 ggagttcaag ttcaccctgt acaagaacga cctgctgctg gtgaaggaca ccgagaccaa
     4441 ggagcagcag ctgttccgct tcctgtcgcg caccatgccg aagcagaagc actacgtgga
     4501 gctgaagccg tacgacaagc agaagttcga gggcggcgag gccctgatca aggtgctggg
     4561 caacgtggcc aactcgggcc agtgcaagaa gggcctgggc aagtcgaaca tctcgatcta
     4621 caaggtgcgc accgacgtgc tgggcaacca gcacatcatc aagaacgagg gcgacaagcc
     4681 gaagctggac ttctaaatac tctggccatt tttctttacc gcctttgagt gagctgatac
     4741 cgctcgccgc agccgaacga ccgagcgcaa cgcgtgcggc cgctgattag ctaagcagaa
     4801 ggccatcctg acggatggcc tttttctaga aaaaaaaaaa gcgccgcaac tgcggcgctt
     4861 tttttttttt aattaagtga tagattgcga tgcgtgtgct cggttgatat aatctgtcag
     4921 atcaggagga cttcgcatgt cgcggttgga taagtccaag gtcatcaact cggcgctgga
     4981 gctgctgaac gaggtcggta tcgagggact gaccacgcgc aagttggccc aaaagctcgg
     5041 cgtggaacaa ccgaccctct actggcacgt gaagaacaag cgcgcgctgc tggatgcgtt
     5101 ggccatcgag atgctggatc gccatcacac gcacttttgc cccctggagg gcgagagctg
     5161 gcaggatttt ctgcggaaca acgcgaagtc gttccggtgc gccctcctga gccatcggga
     5221 tggcgccaaa gtccacttgg gcacccgccc gaccgagaag caatacgaga ccctggagaa
     5281 ccagctggcc ttcctctgtc aacagggttt ttcgttggag aacgccctgt atgccctgtc
     5341 cgccgtggga cacttcacct tgggctgcgt gttggaggat caggaacatc aggtggcgaa
     5401 agaggaacgg gagaccccca cgacggattc gatgcccccg ttgttgcgcc aggcgatcga
     5461 actgtttgac caccagggcg ccgaacccgc ctttttgttc gggctggagc tgatcatctg
     5521 cgggctggaa aagcaactga agtgcgaaag cggttcgtga taacgccgaa aaccccgctt
     5581 cggcggggtt ttgccgcggt ctgcgttgtc gggaagattt aaaataaaaa aggggacctc
     5641 tagggtcccc aattaaatag ttaacactag tattaaaaaa aaaagccccg cgattgcggg
     5701 gctttttttt ttcgacggcg agggttccga ccgctgcaac tcccggtgca accttgtccc
     5761 ggtctattct cttcactgca ccagctccaa tctggtgtga atgcccctcg tctgttcgcg
     5821 caggcggggg gctctattcg tttgtcagca tcgaaagtag ccagatcagg gatgcgttgc
     5881 aaccgcgtat gcccaggtca gaagagtcgc acaagagttg cagacccctg gaaagaaaaa
     5941 tggccagagg gcgaaaacac cctctgacca gcggagcggg cgacgggaat cgaacccgcg
     6001 tagctagttt ggaagaatgg gtgtctgccg accacatata tgggccggtc aagataggtt
     6061 tttaccccct ctcggctgca tcctctaagt ggaaagaaat tgcaggtcgt agaagcgcgt
     6121 tgaagcctga gagttgcaca ggagttgcaa cccggtagcc ttgttcacga cgagaggaga
     6181 cctagttggc acgtcgcgga tggggatcgc tgaagactca gcgcagcggg aggatccaag
     6241 cctcatacgt caacccgcag gacggtgtga ggtactacgc gctgcagacc tacgacaaca
     6301 agatggacgc cgaagcctgg ctcgcgggcg agaagcggct catcgagatg gagacctgga
     6361 cccctccaca ggaccgggcg aagaaggcag ccgccagcgc catcacgctg gaggagtaca
     6421 cccggaagtg gctcgtggag cgcgacctcg cagacggcac cagggatctg tacagcgggc
     6481 acgcggagcg ccgcatctac ccggtgctag gtgaagtggc ggtcacagag atgacgccag
     6541 ctctggtgcg tgcgtggtgg gccgggatgg gtaggaagca cccgactgcc cgccggcatg
     6601 cctacaacgt cctccgggcg gtgatgaaca cagcggtcga ggacaagctg atcgcagaga
     6661 acccgtgccg gatcgagcag aaggcagccg atgagcgcga cgtagaggcg ctgacgcctg
     6721 aggagctgga catcgtcgcc gctgagatct tcgtgcacta ccggatcgcg gcatacatcc
     6781 tggcgtggac gagcctccgg ttcggagagc tgatcgagct tcgccgcaag gacatcgtgg
     6841 acgacggcat gacgatgaag ctccgggtgc gccgtggcgc ttcccgcgtg gggaacaaga
     6901 tcgtcgttgg caacgccaag accgtccggt cgaagcgtcc tgtgacggtt ccgcctcacg
     6961 tcgcggagat gatccgagcg cacatgaagg accgtacgaa gatgaacaag ggccccgagg
     7021 cattcctggt gaccacgacg cagggcaacc ggctgtcgaa gtccgcgttc accaagtcgc
     7081 tgaagcgtgg ctacgccaag atcggtcggc cggaactccg catccacgac ctccgcgctg
     7141 tcggcgctac gttcgccgct caggcaggtg cgacgaccaa ggagctgatg gcccgcctcg
     7201 gtcacacgac tcctaggatg gcgatgaagt accagatggc gtctgaggcc cgcgacgagg
     7261 ctatcgctga ggcgatgtcc aagctggcca agacctcctg aaacgcaaaa agcccccctc
     7321 ccaaggacac tgagtcctaa agaggggggt ttcttgtcag tacgcgaaga accacgcctg
     7381 gccgcgagcg ccagcaccgc cgctctgtgc ggagacctgg gcaccagccc cgccgccgcc
     7441 aggagcattg ccgttcccgc cagctagcca cctgacgtcg gggggggggg aaagccacgt
     7501 tgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     7561 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     7621 aacgtcttgc tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     7681 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     7741 ccatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     7801 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     7861 tatccgtact cctgatgatg catggttact caccactgcg atccccggga aaacagcatt
     7921 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     7981 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     8041 tcgcctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     8101 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata atcttttgcc
     8161 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     8221 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     8281 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     8341 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     8401 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     8461 gacttgacgg gacggcggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag
     8521 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc
     8581 aactggtcca cctacaacaa agctctcacc aaccgtggct ccctcacgat atc
//
