LOCUS       sequence-483385-        8634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi gene silencing plasmid with sgRNA (Rv1259) 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 8634)
  TITLE     Rhee Lab CRISPRi plasmids
REFERENCE   2  (bases 1 to 8634)
  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_Rv1259
FEATURES             Location/Qualifiers
     source          1..8634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(499..1087)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(579..598)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1270..1288
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1300..1318
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      4798..4825
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(7591..7610)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7602..8417
                     /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(7675..7694)
                     /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 tctgggaatc ctgcatccgg
      181 gctgcggcag cgtttttgta ctcgaaagaa gctacaaaga taaggcttca tgccgaaatc
      241 aacaccctgt cattttatgg cagggtgttt tttttttgtc gacttgggga ccctagaggt
      301 cccctttttt atttttttgg atgaagagca ttatcaatgg ctcttcacag gaagcttaaa
      361 aaaaaaagcc ccgcgattgc ggggcttttt ttttttaata cggttatcca cagaatcagg
      421 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      481 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
      541 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      601 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      661 ctttctcccc tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
      721 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      781 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      841 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
      901 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
      961 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1021 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1081 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcatggaa caataaacga
     1141 aaggctcagt cgaaagactg ggcctttcgt ttatctgttg tttgaaaaaa aaaagccccg
     1201 cgattgcggg gctttttttt ttcatatgtc tgaccaggga aaatagccct ctgacctggg
     1261 gatttgactt ccctatcagt gatagagata atctgggagt ccctatcagt gatagagaac
     1321 gcggtatcga tatgtcggac ctggtgctgg gcctggccat cggcatcggc tcggtgggcg
     1381 tgggcatcct gaacaaggtg accggcgaga tcatccacaa gaactcgcgc atcttcccgg
     1441 ccgcccaggc cgagaacaac ctggtgcgcc gcaccaaccg ccagggccgc cgcctggccc
     1501 gccgcaagaa gcaccgccgc gtgcgcctga accgcctgtt cgaggagtcg ggcctgatca
     1561 ccgacttcac caagatctcg atcaacctga acccgtacca gctgcgcgtg aagggcctga
     1621 ccgacgagct gtcgaacgag gagctgttca tcgccctgaa gaacatggtg aagcaccgcg
     1681 gcatctcgta cctggacgac gcctcggacg acggcaactc gtcggtgggc gactacgccc
     1741 agatcgtgaa ggagaactcg aagcagctgg agaccaagac cccgggccag atccagctgg
     1801 agcgctacca gacctacggc cagctgcgcg gcgacttcac cgtggagaag gacggcaaga
     1861 agcaccgcct gatcaacgtg ttcccgacct cggcctaccg ctcggaggcc ctgcgcatcc
     1921 tgcagaccca gcaggagttc aacccgcaga tcaccgacga gttcatcaac cgctacctgg
     1981 agatcctgac cggcaagcgc aagtactacc acggcccggg caacgagaag tcgcgcaccg
     2041 actacggccg ctaccgcacc tcgggcgaga ccctggacaa catcttcggc atcctgatcg
     2101 gcaagtgcac cttctacccg gacgagttcc gcgccgccaa ggcctcgtac accgcccagg
     2161 agttcaacct gctgaacgac ctgaacaacc tgaccgtgcc gaccgagacc aagaagctgt
     2221 cgaaggagca gaagaaccag atcatcaact acgtgaagaa cgagaaggcc atgggcccgg
     2281 ccaagctgtt caagtacatc gccaagctgc tgtcgtgcga cgtggccgac atcaagggct
     2341 accgcatcga caagtcgggc aaggccgaga tccacacctt cgaggcctac cgcaagatga
     2401 agaccctgga gaccctggac atcgagcaga tggaccgcga gaccctggac aagctggcct
     2461 acgtgctgac cctgaacacc gagcgcgagg gcatccagga ggccctggag cacgagttcg
     2521 ccgacggctc gttctcgcag aagcaggtgg acgagctggt gcagttccgc aaggccaact
     2581 cgtcgatctt cggcaagggc tggcacaact tctcggtgaa gctgatgatg gagctgatcc
     2641 cggagctgta cgagacctcg gaggagcaga tgaccatcct gacccgcctg ggcaagcaga
     2701 agaccacctc gtcgtcgaac aagaccaagt acatcgacga gaagctgctg accgaggaga
     2761 tctacaaccc ggtggtggcc aagtcggtgc gccaggccat caagatcgtg aacgccgcca
     2821 tcaaggagta cggcgacttc gacaacatcg tgatcgagat ggcccgcgag accaacgagg
     2881 acgacgagaa gaaggccatc cagaagatcc agaaggccaa caaggacgag aaggacgccg
     2941 ccatgctgaa ggccgccaac cagtacaacg gcaaggccga gctgccgcac tcggtgttcc
     3001 acggccacaa gcagctggcc accaagatcc gcctgtggca ccagcagggc gagcgctgcc
     3061 tgtacaccgg caagaccatc tcgatccacg acctgatcaa caactcgaac cagttcgagg
     3121 tggacgccat cctgccgctg tcgatcacct tcgacgactc gctggccaac aaggtgctgg
     3181 tgtacgccac cgccaaccag gagaagggcc agcgcacccc gtaccaggcc ctggactcga
     3241 tggacgacgc ctggtcgttc cgcgagctga aggccttcgt gcgcgagtcg aagaccctgt
     3301 cgaacaagaa gaaggagtac ctgctgaccg aggaggacat ctcgaagttc gacgtgcgca
     3361 agaagttcat cgagcgcaac ctggtggaca cccgctacgc ctcgcgcgtg gtgctgaacg
     3421 ccctgcagga gcacttccgc gcccacaaga tcgacaccaa ggtgtcggtg gtgcgcggcc
     3481 agttcacctc gcagctgcgc cgccactggg gcatcgagaa gacccgcgac acctaccacc
     3541 accacgccgt ggacgccctg atcatcgccg cctcgtcgca gctgaacctg tggaagaagc
     3601 agaagaacac cctggtgtcg tactcggagg accagctgct ggacatcgag accggcgagc
     3661 tgatctcgga cgacgagtac aaggagtcgg tgttcaaggc cccgtaccag cacttcgtgg
     3721 acaccctgaa gtcgaaggag ttcgaggact cgatcctgtt ctcgtaccag gtggactcga
     3781 agttcaaccg caagatctcg gacgccacca tctacgccac ccgccaggcc aaggtgggca
     3841 aggacaaggc cgacgagacc tacgtgctgg gcaagatcaa ggacatctac acccaggacg
     3901 gctacgacgc cttcatgaag atctacaaga aggacaagtc gaagttcctg atgtaccgcc
     3961 acgacccgca gaccttcgag aaggtgatcg agccgatcct ggagaactac ccgaacaagc
     4021 agatcaacga caagggcaag gaggtgccgt gcaacccgtt cctgaagtac aaggaggagc
     4081 acggctacat ccgcaagtac tcgaagaagg gcaacggccc ggagatcaag tcgctgaagt
     4141 actacgactc gaagctgggc aaccacatcg acatcacccc gaaggactcg aacaacaagg
     4201 tggtgctgca gtcggtgtcg ccgtggcgcg ccgacgtgta cttcaacaag accaccggca
     4261 agtacgagat cctgggcctg aagtacgccg acctgcagtt cgacaagggc accggcacct
     4321 acaagatctc gcaggagaag tacaacgaca tcaagaagaa ggagggcgtg gactcggact
     4381 cggagttcaa gttcaccctg tacaagaacg acctgctgct ggtgaaggac accgagacca
     4441 aggagcagca gctgttccgc ttcctgtcgc gcaccatgcc gaagcagaag cactacgtgg
     4501 agctgaagcc gtacgacaag cagaagttcg agggcggcga ggccctgatc aaggtgctgg
     4561 gcaacgtggc caactcgggc cagtgcaaga agggcctggg caagtcgaac atctcgatct
     4621 acaaggtgcg caccgacgtg ctgggcaacc agcacatcat caagaacgag ggcgacaagc
     4681 cgaagctgga cttctaaata ctctggccat ttttctttac cgcctttgag tgagctgata
     4741 ccgctcgccg cagccgaacg accgagcgca acgcgtgcgg ccgctgatta gctaagcaga
     4801 aggccatcct gacggatggc ctttttctag aaaaaaaaaa agcgccgcaa ctgcggcgct
     4861 tttttttttt taattaagtg atagattgcg atgcgtgtgc tcggttgata taatctgtca
     4921 gatcaggagg acttcgcatg tcgcggttgg ataagtccaa ggtcatcaac tcggcgctgg
     4981 agctgctgaa cgaggtcggt atcgagggac tgaccacgcg caagttggcc caaaagctcg
     5041 gcgtggaaca accgaccctc tactggcacg tgaagaacaa gcgcgcgctg ctggatgcgt
     5101 tggccatcga gatgctggat cgccatcaca cgcacttttg ccccctggag ggcgagagct
     5161 ggcaggattt tctgcggaac aacgcgaagt cgttccggtg cgccctcctg agccatcggg
     5221 atggcgccaa agtccacttg ggcacccgcc cgaccgagaa gcaatacgag accctggaga
     5281 accagctggc cttcctctgt caacagggtt tttcgttgga gaacgccctg tatgccctgt
     5341 ccgccgtggg acacttcacc ttgggctgcg tgttggagga tcaggaacat caggtggcga
     5401 aagaggaacg ggagaccccc acgacggatt cgatgccccc gttgttgcgc caggcgatcg
     5461 aactgtttga ccaccagggc gccgaacccg cctttttgtt cgggctggag ctgatcatct
     5521 gcgggctgga aaagcaactg aagtgcgaaa gcggttcgtg ataacgccga aaaccccgct
     5581 tcggcggggt tttgccgcgg tctgcgttgt cgggaagatt taaaataaaa aaggggacct
     5641 ctagggtccc caattaaata gttaacacta gtattaaaaa aaaaagcccc gcgattgcgg
     5701 ggcttttttt tttcgacggc gagggttccg accgctgcaa ctcccggtgc aaccttgtcc
     5761 cggtctattc tcttcactgc accagctcca atctggtgtg aatgcccctc gtctgttcgc
     5821 gcaggcgggg ggctctattc gtttgtcagc atcgaaagta gccagatcag ggatgcgttg
     5881 caaccgcgta tgcccaggtc agaagagtcg cacaagagtt gcagacccct ggaaagaaaa
     5941 atggccagag ggcgaaaaca ccctctgacc agcggagcgg gcgacgggaa tcgaacccgc
     6001 gtagctagtt tggaagaatg ggtgtctgcc gaccacatat atgggccggt caagataggt
     6061 ttttaccccc tctcggctgc atcctctaag tggaaagaaa ttgcaggtcg tagaagcgcg
     6121 ttgaagcctg agagttgcac aggagttgca acccggtagc cttgttcacg acgagaggag
     6181 acctagttgg cacgtcgcgg atggggatcg ctgaagactc agcgcagcgg gaggatccaa
     6241 gcctcatacg tcaacccgca ggacggtgtg aggtactacg cgctgcagac ctacgacaac
     6301 aagatggacg ccgaagcctg gctcgcgggc gagaagcggc tcatcgagat ggagacctgg
     6361 acccctccac aggaccgggc gaagaaggca gccgccagcg ccatcacgct ggaggagtac
     6421 acccggaagt ggctcgtgga gcgcgacctc gcagacggca ccagggatct gtacagcggg
     6481 cacgcggagc gccgcatcta cccggtgcta ggtgaagtgg cggtcacaga gatgacgcca
     6541 gctctggtgc gtgcgtggtg ggccgggatg ggtaggaagc acccgactgc ccgccggcat
     6601 gcctacaacg tcctccgggc ggtgatgaac acagcggtcg aggacaagct gatcgcagag
     6661 aacccgtgcc ggatcgagca gaaggcagcc gatgagcgcg acgtagaggc gctgacgcct
     6721 gaggagctgg acatcgtcgc cgctgagatc ttcgtgcact accggatcgc ggcatacatc
     6781 ctggcgtgga cgagcctccg gttcggagag ctgatcgagc ttcgccgcaa ggacatcgtg
     6841 gacgacggca tgacgatgaa gctccgggtg cgccgtggcg cttcccgcgt ggggaacaag
     6901 atcgtcgttg gcaacgccaa gaccgtccgg tcgaagcgtc ctgtgacggt tccgcctcac
     6961 gtcgcggaga tgatccgagc gcacatgaag gaccgtacga agatgaacaa gggccccgag
     7021 gcattcctgg tgaccacgac gcagggcaac cggctgtcga agtccgcgtt caccaagtcg
     7081 ctgaagcgtg gctacgccaa gatcggtcgg ccggaactcc gcatccacga cctccgcgct
     7141 gtcggcgcta cgttcgccgc tcaggcaggt gcgacgacca aggagctgat ggcccgcctc
     7201 ggtcacacga ctcctaggat ggcgatgaag taccagatgg cgtctgaggc ccgcgacgag
     7261 gctatcgctg aggcgatgtc caagctggcc aagacctcct gaaacgcaaa aagcccccct
     7321 cccaaggaca ctgagtccta aagagggggg tttcttgtca gtacgcgaag aaccacgcct
     7381 ggccgcgagc gccagcaccg ccgctctgtg cggagacctg ggcaccagcc ccgccgccgc
     7441 caggagcatt gccgttcccg ccagctagcc acctgacgtc gggggggggg gaaagccacg
     7501 ttgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     7561 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     7621 aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     7681 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     7741 cccatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     7801 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     7861 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat
     7921 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     7981 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     8041 ttcgcctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     8101 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aatcttttgc
     8161 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     8221 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     8281 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     8341 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     8401 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc
     8461 tgacttgacg ggacggcggc tttgttgaat aaatcgaact tttgctgagt tgaaggatca
     8521 gatcacgcat cttcccgaca acgcagaccg ttccgtggca aagcaaaagt tcaaaatcac
     8581 caactggtcc acctacaaca aagctctcac caaccgtggc tccctcacga tatc
//
