LOCUS       sequence-237528-        6863 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TetR_tetR/tetA promoters_dCas9-CP_CM_P15A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6863)
  AUTHORS   Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, 
            Savage DF
  TITLE     Profiling of engineering hotspots identifies an allosteric 
            CRISPR-Cas9 switch.
  JOURNAL   Nat Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528.
  PUBMED    27136077
REFERENCE   2  (bases 1 to 6863)
  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"; journalName: "Nat 
            Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528."
COMMENT     Sequence Label: pBLO 9.16_bCas9-CP1282
FEATURES             Location/Qualifiers
     source          1..6863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(7..633)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     538..557
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        649..704
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    685..703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      5028..5099
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5115..5142
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5170..5187)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5304..5849)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(5384..5403)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5963..6057)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6078..6737)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6652..6671
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6738..6840)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
       61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
      121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
      181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
      241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
      301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
      361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
      421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
      481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
      541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
      601 aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta
      661 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc
      721 taggatccgt tttcgcattt atcgtgaaac gctttcgcat tttgagtgaa ttatatgata
      781 gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga caaaccaata
      841 cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct
      901 gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt
      961 ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg
     1021 agtcagctag gaggtgacgg cggatcagga ggctctggcg gttcaggtgg atcaggcggt
     1081 agcggaggtt caggtggtat ggataagaaa tactcaatag gcttagctat cggcacaaat
     1141 agcgtcggat gggcggtgat cactgatgaa tataaggttc cgtctaaaaa gttcaaggtt
     1201 ctgggaaata cagaccgcca cagtatcaaa aaaaatctta taggggctct tttatttgac
     1261 agtggagaga cagcggaagc gactcgtctc aaacggacag ctcgtagaag gtatacacgt
     1321 cggaagaatc gtatttgtta tctacaggag attttttcaa atgagatggc gaaagtagat
     1381 gatagtttct ttcatcgact tgaagagtct tttttggtgg aagaagacaa gaagcatgaa
     1441 cgtcatccta tttttggaaa tatagtagat gaagttgctt atcatgagaa atatccaact
     1501 atctatcatc tgcgaaaaaa attggtagat tctactgata aagcggattt gcgcttaatc
     1561 tatttggcct tagcgcatat gattaagttt cgtggtcatt ttttgattga gggagattta
     1621 aatcctgata atagtgatgt ggacaaacta tttatccagt tggtacaaac ctacaatcaa
     1681 ttatttgaag aaaaccctat taacgcaagt ggagtagatg ctaaagcgat tctttctgca
     1741 cgattgagta aatcaagacg attagaaaat ctcattgctc agctccccgg tgagaagaaa
     1801 aatggcttat ttgggaatct cattgctttg tcattgggtt tgacccctaa ttttaaatca
     1861 aattttgatt tggcagaaga tgctaaatta cagctttcaa aagatactta cgatgatgat
     1921 ttagataatt tattggcgca aattggagat caatatgctg atttgttttt ggcagctaag
     1981 aatttatcag atgctatttt actttcagat atcctaagag taaatactga aataactaag
     2041 gctcccctat cagcttcaat gattaaacgc tacgatgaac atcatcaaga cttgactctt
     2101 ttaaaagctt tagttcgaca acaacttcca gaaaagtata aagaaatctt ttttgatcaa
     2161 tcaaaaaacg gatatgcagg ttatattgat gggggagcta gccaagaaga attttataaa
     2221 tttatcaaac caattttaga aaaaatggat ggtactgagg aattattggt gaaactaaat
     2281 cgtgaagatt tgctgcgcaa gcaacggacc tttgacaacg gctctattcc ccatcaaatt
     2341 cacttgggtg agctgcatgc tattttgaga agacaagaag acttttatcc atttttaaaa
     2401 gacaatcgtg agaagattga aaaaatcttg acttttcgaa ttccttatta tgttggtcca
     2461 ttggcgcgtg gcaatagtcg ttttgcatgg atgactcgga agtctgaaga aacaattacc
     2521 ccatggaatt ttgaagaagt tgtcgataaa ggtgcttcag ctcaatcatt tattgaacgc
     2581 atgacaaact ttgataaaaa tcttccaaat gaaaaagtac taccaaaaca tagtttgctt
     2641 tatgagtatt ttacggttta taacgaattg acaaaggtca aatatgttac tgaaggaatg
     2701 cgaaaaccag catttctttc aggtgaacag aagaaagcca ttgttgattt actcttcaaa
     2761 acaaatcgaa aagtaaccgt taagcaatta aaagaagatt atttcaaaaa aatagaatgt
     2821 tttgatagtg ttgaaatttc aggagttgaa gatagattta atgcttcatt aggtacctac
     2881 catgatttgc taaaaattat taaagataaa gattttttgg ataatgaaga aaatgaagat
     2941 atcttagagg atattgtttt aacattgacc ttatttgaag atagggagat gattgaggaa
     3001 agacttaaaa catatgctca cctctttgat gataaggtga tgaaacagct taaacgtcgc
     3061 cgttatactg gttggggacg tttgtctcga aaattgatta atggtattag ggataagcaa
     3121 tctggcaaaa caatattaga ttttttgaaa tcagatggtt ttgccaatcg caattttatg
     3181 cagctgatcc atgatgatag tttgacattt aaagaagaca ttcaaaaagc acaagtgtct
     3241 ggacaaggcg atagtttaca tgaacatatt gcaaatttag ctggtagccc tgctattaaa
     3301 aaaggtattt tacagactgt aaaagttgtt gatgaattgg tcaaagtaat ggggcggcat
     3361 aagccagaaa atatcgttat tgaaatggca cgtgaaaatc agacaactca aaagggccag
     3421 aaaaattcgc gagagcgtat gaaacgaatc gaagaaggta tcaaagaatt aggaagtcag
     3481 attcttaaag agcatcctgt tgaaaatact caattgcaaa atgaaaagct ctatctctat
     3541 tatctccaaa atggaagaga catgtatgtg gaccaagaat tagatattaa tcgtttaagt
     3601 gattatgatg tcgatgccat tgttccacaa agtttcctta aagacgattc aatagacaat
     3661 aaggtcttaa cgcgttctga taaaaatcgt ggtaaatcgg ataacgttcc aagtgaagaa
     3721 gtagtcaaaa agatgaaaaa ctattggaga caacttctaa acgccaagtt aatcactcaa
     3781 cgtaagtttg ataatttaac gaaagctgaa cgtggaggtt tgagtgaact tgataaagct
     3841 ggttttatca aacgccaatt ggttgaaact cgccaaatca ctaagcatgt ggcacaaatt
     3901 ttggatagtc gcatgaatac taaatacgat gaaaatgata aacttattcg agaggttaaa
     3961 gtgattacct taaaatctaa attagtttct gacttccgaa aagatttcca attctataaa
     4021 gtacgtgaga ttaacaatta ccatcatgcc catgatgcgt atctaaatgc cgtcgttgga
     4081 actgctttga ttaagaaata tccaaaactt gaatcggagt ttgtctatgg tgattataaa
     4141 gtttatgatg ttcgtaaaat gattgctaag tctgagcaag aaataggcaa agcaaccgca
     4201 aaatatttct tttactctaa tatcatgaac ttcttcaaaa cagaaattac acttgcaaat
     4261 ggagagattc gcaaacgccc tctaatcgaa actaatgggg aaactggaga aattgtctgg
     4321 gataaagggc gagattttgc cacagtgcgc aaagtattgt ccatgcccca agtcaatatt
     4381 gtcaagaaaa cagaagtaca gacaggcgga ttctccaagg agtcaatttt accaaaaaga
     4441 aattcggaca agcttattgc tcgtaaaaaa gactgggatc caaaaaaata tggtggtttt
     4501 gatagtccaa cggtagctta ttcagtccta gtggttgcta aggtggaaaa agggaaatcg
     4561 aagaagttaa aatccgttaa agagttacta gggatcacaa ttatggaaag aagttccttt
     4621 gaaaaaaatc cgattgactt tttagaagct aaaggatata aggaagttaa aaaagactta
     4681 atcattaaac tacctaaata tagtcttttt gagttagaaa acggtcgtaa acggatgctg
     4741 gctagtgccg gagaattaca aaaaggaaat gagctggctc tgccaagcaa atatgtgaat
     4801 tttttatatt tagctagtca ttatgaaaag ttgaagggta gtccagaaga taacgaacaa
     4861 aaacaattgt ttgtggagca gcataagcat tatttagatg agattattga gcaaatcagt
     4921 gaattttcta agcgtgttat tttagcatga ttgattgaac gaaaaacgcg aaagcgtttc
     4981 acgataaatg cgaaaacgga tcctctcgag taaggatctc caggcatcaa ataaaacgaa
     5041 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct
     5101 actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatacc tagggatata
     5161 ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat
     5221 ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt
     5281 gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa
     5341 atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5401 ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt
     5461 ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt
     5521 tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc
     5581 cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc
     5641 cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg
     5701 aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg
     5761 tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag
     5821 agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt
     5881 tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat
     5941 ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg
     6001 ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa
     6061 gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca
     6121 ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc
     6181 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     6241 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     6301 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     6361 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     6421 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     6481 tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc
     6541 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     6601 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     6661 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     6721 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat
     6781 acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca
     6841 acgtctcatt ttcgccagat atc
//
