LOCUS       sequence-483380-        8632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi gene silencing plasmid with sgRNA (Rv1420) 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 8632)
  TITLE     Rhee Lab CRISPRi plasmids
REFERENCE   2  (bases 1 to 8632)
  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_Rv1420
FEATURES             Location/Qualifiers
     source          1..8632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(497..1085)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(577..596)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1268..1286
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1298..1316
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      4796..4823
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(7589..7608)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7600..8415
                     /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(7673..7692)
                     /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 tctgggagaa tctacgcgac
      181 ttcctttccg tttttgtact cgaaagaagc tacaaagata aggcttcatg ccgaaatcaa
      241 caccctgtca ttttatggca gggtgttttt tttttgtcga cttggggacc ctagaggtcc
      301 ccttttttat ttttttggat gaagagcatt atcaatggct cttcacagga agcttaaaaa
      361 aaaaagcccc gcgattgcgg ggcttttttt ttttaatacg gttatccaca gaatcagggg
      421 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
      481 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
      541 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
      601 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
      661 ttctcccctc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
      721 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
      781 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
      841 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
      901 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
      961 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1021 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1081 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcatggaaca ataaacgaaa
     1141 ggctcagtcg aaagactggg cctttcgttt atctgttgtt tgaaaaaaaa aagccccgcg
     1201 attgcggggc tttttttttt catatgtctg accagggaaa atagccctct gacctgggga
     1261 tttgacttcc ctatcagtga tagagataat ctgggagtcc ctatcagtga tagagaacgc
     1321 ggtatcgata tgtcggacct ggtgctgggc ctggccatcg gcatcggctc ggtgggcgtg
     1381 ggcatcctga acaaggtgac cggcgagatc atccacaaga actcgcgcat cttcccggcc
     1441 gcccaggccg agaacaacct ggtgcgccgc accaaccgcc agggccgccg cctggcccgc
     1501 cgcaagaagc accgccgcgt gcgcctgaac cgcctgttcg aggagtcggg cctgatcacc
     1561 gacttcacca agatctcgat caacctgaac ccgtaccagc tgcgcgtgaa gggcctgacc
     1621 gacgagctgt cgaacgagga gctgttcatc gccctgaaga acatggtgaa gcaccgcggc
     1681 atctcgtacc tggacgacgc ctcggacgac ggcaactcgt cggtgggcga ctacgcccag
     1741 atcgtgaagg agaactcgaa gcagctggag accaagaccc cgggccagat ccagctggag
     1801 cgctaccaga cctacggcca gctgcgcggc gacttcaccg tggagaagga cggcaagaag
     1861 caccgcctga tcaacgtgtt cccgacctcg gcctaccgct cggaggccct gcgcatcctg
     1921 cagacccagc aggagttcaa cccgcagatc accgacgagt tcatcaaccg ctacctggag
     1981 atcctgaccg gcaagcgcaa gtactaccac ggcccgggca acgagaagtc gcgcaccgac
     2041 tacggccgct accgcacctc gggcgagacc ctggacaaca tcttcggcat cctgatcggc
     2101 aagtgcacct tctacccgga cgagttccgc gccgccaagg cctcgtacac cgcccaggag
     2161 ttcaacctgc tgaacgacct gaacaacctg accgtgccga ccgagaccaa gaagctgtcg
     2221 aaggagcaga agaaccagat catcaactac gtgaagaacg agaaggccat gggcccggcc
     2281 aagctgttca agtacatcgc caagctgctg tcgtgcgacg tggccgacat caagggctac
     2341 cgcatcgaca agtcgggcaa ggccgagatc cacaccttcg aggcctaccg caagatgaag
     2401 accctggaga ccctggacat cgagcagatg gaccgcgaga ccctggacaa gctggcctac
     2461 gtgctgaccc tgaacaccga gcgcgagggc atccaggagg ccctggagca cgagttcgcc
     2521 gacggctcgt tctcgcagaa gcaggtggac gagctggtgc agttccgcaa ggccaactcg
     2581 tcgatcttcg gcaagggctg gcacaacttc tcggtgaagc tgatgatgga gctgatcccg
     2641 gagctgtacg agacctcgga ggagcagatg accatcctga cccgcctggg caagcagaag
     2701 accacctcgt cgtcgaacaa gaccaagtac atcgacgaga agctgctgac cgaggagatc
     2761 tacaacccgg tggtggccaa gtcggtgcgc caggccatca agatcgtgaa cgccgccatc
     2821 aaggagtacg gcgacttcga caacatcgtg atcgagatgg cccgcgagac caacgaggac
     2881 gacgagaaga aggccatcca gaagatccag aaggccaaca aggacgagaa ggacgccgcc
     2941 atgctgaagg ccgccaacca gtacaacggc aaggccgagc tgccgcactc ggtgttccac
     3001 ggccacaagc agctggccac caagatccgc ctgtggcacc agcagggcga gcgctgcctg
     3061 tacaccggca agaccatctc gatccacgac ctgatcaaca actcgaacca gttcgaggtg
     3121 gacgccatcc tgccgctgtc gatcaccttc gacgactcgc tggccaacaa ggtgctggtg
     3181 tacgccaccg ccaaccagga gaagggccag cgcaccccgt accaggccct ggactcgatg
     3241 gacgacgcct ggtcgttccg cgagctgaag gccttcgtgc gcgagtcgaa gaccctgtcg
     3301 aacaagaaga aggagtacct gctgaccgag gaggacatct cgaagttcga cgtgcgcaag
     3361 aagttcatcg agcgcaacct ggtggacacc cgctacgcct cgcgcgtggt gctgaacgcc
     3421 ctgcaggagc acttccgcgc ccacaagatc gacaccaagg tgtcggtggt gcgcggccag
     3481 ttcacctcgc agctgcgccg ccactggggc atcgagaaga cccgcgacac ctaccaccac
     3541 cacgccgtgg acgccctgat catcgccgcc tcgtcgcagc tgaacctgtg gaagaagcag
     3601 aagaacaccc tggtgtcgta ctcggaggac cagctgctgg acatcgagac cggcgagctg
     3661 atctcggacg acgagtacaa ggagtcggtg ttcaaggccc cgtaccagca cttcgtggac
     3721 accctgaagt cgaaggagtt cgaggactcg atcctgttct cgtaccaggt ggactcgaag
     3781 ttcaaccgca agatctcgga cgccaccatc tacgccaccc gccaggccaa ggtgggcaag
     3841 gacaaggccg acgagaccta cgtgctgggc aagatcaagg acatctacac ccaggacggc
     3901 tacgacgcct tcatgaagat ctacaagaag gacaagtcga agttcctgat gtaccgccac
     3961 gacccgcaga ccttcgagaa ggtgatcgag ccgatcctgg agaactaccc gaacaagcag
     4021 atcaacgaca agggcaagga ggtgccgtgc aacccgttcc tgaagtacaa ggaggagcac
     4081 ggctacatcc gcaagtactc gaagaagggc aacggcccgg agatcaagtc gctgaagtac
     4141 tacgactcga agctgggcaa ccacatcgac atcaccccga aggactcgaa caacaaggtg
     4201 gtgctgcagt cggtgtcgcc gtggcgcgcc gacgtgtact tcaacaagac caccggcaag
     4261 tacgagatcc tgggcctgaa gtacgccgac ctgcagttcg acaagggcac cggcacctac
     4321 aagatctcgc aggagaagta caacgacatc aagaagaagg agggcgtgga ctcggactcg
     4381 gagttcaagt tcaccctgta caagaacgac ctgctgctgg tgaaggacac cgagaccaag
     4441 gagcagcagc tgttccgctt cctgtcgcgc accatgccga agcagaagca ctacgtggag
     4501 ctgaagccgt acgacaagca gaagttcgag ggcggcgagg ccctgatcaa ggtgctgggc
     4561 aacgtggcca actcgggcca gtgcaagaag ggcctgggca agtcgaacat ctcgatctac
     4621 aaggtgcgca ccgacgtgct gggcaaccag cacatcatca agaacgaggg cgacaagccg
     4681 aagctggact tctaaatact ctggccattt ttctttaccg cctttgagtg agctgatacc
     4741 gctcgccgca gccgaacgac cgagcgcaac gcgtgcggcc gctgattagc taagcagaag
     4801 gccatcctga cggatggcct ttttctagaa aaaaaaaaag cgccgcaact gcggcgcttt
     4861 ttttttttta attaagtgat agattgcgat gcgtgtgctc ggttgatata atctgtcaga
     4921 tcaggaggac ttcgcatgtc gcggttggat aagtccaagg tcatcaactc ggcgctggag
     4981 ctgctgaacg aggtcggtat cgagggactg accacgcgca agttggccca aaagctcggc
     5041 gtggaacaac cgaccctcta ctggcacgtg aagaacaagc gcgcgctgct ggatgcgttg
     5101 gccatcgaga tgctggatcg ccatcacacg cacttttgcc ccctggaggg cgagagctgg
     5161 caggattttc tgcggaacaa cgcgaagtcg ttccggtgcg ccctcctgag ccatcgggat
     5221 ggcgccaaag tccacttggg cacccgcccg accgagaagc aatacgagac cctggagaac
     5281 cagctggcct tcctctgtca acagggtttt tcgttggaga acgccctgta tgccctgtcc
     5341 gccgtgggac acttcacctt gggctgcgtg ttggaggatc aggaacatca ggtggcgaaa
     5401 gaggaacggg agacccccac gacggattcg atgcccccgt tgttgcgcca ggcgatcgaa
     5461 ctgtttgacc accagggcgc cgaacccgcc tttttgttcg ggctggagct gatcatctgc
     5521 gggctggaaa agcaactgaa gtgcgaaagc ggttcgtgat aacgccgaaa accccgcttc
     5581 ggcggggttt tgccgcggtc tgcgttgtcg ggaagattta aaataaaaaa ggggacctct
     5641 agggtcccca attaaatagt taacactagt attaaaaaaa aaagccccgc gattgcgggg
     5701 cttttttttt tcgacggcga gggttccgac cgctgcaact cccggtgcaa ccttgtcccg
     5761 gtctattctc ttcactgcac cagctccaat ctggtgtgaa tgcccctcgt ctgttcgcgc
     5821 aggcgggggg ctctattcgt ttgtcagcat cgaaagtagc cagatcaggg atgcgttgca
     5881 accgcgtatg cccaggtcag aagagtcgca caagagttgc agacccctgg aaagaaaaat
     5941 ggccagaggg cgaaaacacc ctctgaccag cggagcgggc gacgggaatc gaacccgcgt
     6001 agctagtttg gaagaatggg tgtctgccga ccacatatat gggccggtca agataggttt
     6061 ttaccccctc tcggctgcat cctctaagtg gaaagaaatt gcaggtcgta gaagcgcgtt
     6121 gaagcctgag agttgcacag gagttgcaac ccggtagcct tgttcacgac gagaggagac
     6181 ctagttggca cgtcgcggat ggggatcgct gaagactcag cgcagcggga ggatccaagc
     6241 ctcatacgtc aacccgcagg acggtgtgag gtactacgcg ctgcagacct acgacaacaa
     6301 gatggacgcc gaagcctggc tcgcgggcga gaagcggctc atcgagatgg agacctggac
     6361 ccctccacag gaccgggcga agaaggcagc cgccagcgcc atcacgctgg aggagtacac
     6421 ccggaagtgg ctcgtggagc gcgacctcgc agacggcacc agggatctgt acagcgggca
     6481 cgcggagcgc cgcatctacc cggtgctagg tgaagtggcg gtcacagaga tgacgccagc
     6541 tctggtgcgt gcgtggtggg ccgggatggg taggaagcac ccgactgccc gccggcatgc
     6601 ctacaacgtc ctccgggcgg tgatgaacac agcggtcgag gacaagctga tcgcagagaa
     6661 cccgtgccgg atcgagcaga aggcagccga tgagcgcgac gtagaggcgc tgacgcctga
     6721 ggagctggac atcgtcgccg ctgagatctt cgtgcactac cggatcgcgg catacatcct
     6781 ggcgtggacg agcctccggt tcggagagct gatcgagctt cgccgcaagg acatcgtgga
     6841 cgacggcatg acgatgaagc tccgggtgcg ccgtggcgct tcccgcgtgg ggaacaagat
     6901 cgtcgttggc aacgccaaga ccgtccggtc gaagcgtcct gtgacggttc cgcctcacgt
     6961 cgcggagatg atccgagcgc acatgaagga ccgtacgaag atgaacaagg gccccgaggc
     7021 attcctggtg accacgacgc agggcaaccg gctgtcgaag tccgcgttca ccaagtcgct
     7081 gaagcgtggc tacgccaaga tcggtcggcc ggaactccgc atccacgacc tccgcgctgt
     7141 cggcgctacg ttcgccgctc aggcaggtgc gacgaccaag gagctgatgg cccgcctcgg
     7201 tcacacgact cctaggatgg cgatgaagta ccagatggcg tctgaggccc gcgacgaggc
     7261 tatcgctgag gcgatgtcca agctggccaa gacctcctga aacgcaaaaa gcccccctcc
     7321 caaggacact gagtcctaaa gaggggggtt tcttgtcagt acgcgaagaa ccacgcctgg
     7381 ccgcgagcgc cagcaccgcc gctctgtgcg gagacctggg caccagcccc gccgccgcca
     7441 ggagcattgc cgttcccgcc agctagccac ctgacgtcgg ggggggggga aagccacgtt
     7501 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     7561 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     7621 acgtcttgct cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     7681 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     7741 catgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     7801 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     7861 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     7921 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     7981 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     8041 cgcctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     8101 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa tcttttgcca
     8161 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     8221 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     8281 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     8341 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     8401 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg
     8461 acttgacggg acggcggctt tgttgaataa atcgaacttt tgctgagttg aaggatcaga
     8521 tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa gcaaaagttc aaaatcacca
     8581 actggtccac ctacaacaaa gctctcacca accgtggctc cctcacgata tc
//
