LOCUS       sequence-238423-        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 9.6_bCas9-CP1010
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 ttatatgaat
      781 ggtgattata aagtttatga tgttcgtaaa atgattgcta agtctgagca agaaataggc
      841 aaagcaaccg caaaatattt cttttactct aatatcatga acttcttcaa aacagaaatt
      901 acacttgcaa atggagagat tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga
      961 gaaattgtct gggataaagg gcgagatttt gccacagtgc gcaaagtatt gtccatgccc
     1021 caagtcaata ttgtcaagaa aacagaagta cagacaggcg gattctccaa ggagtcaatt
     1081 ttaccaaaaa gaaattcgga caagcttatt gctcgtaaaa aagactggga tccaaaaaaa
     1141 tatggtggtt ttgatagtcc aacggtagct tattcagtcc tagtggttgc taaggtggaa
     1201 aaagggaaat cgaagaagtt aaaatccgtt aaagagttac tagggatcac aattatggaa
     1261 agaagttcct ttgaaaaaaa tccgattgac tttttagaag ctaaaggata taaggaagtt
     1321 aaaaaagact taatcattaa actacctaaa tatagtcttt ttgagttaga aaacggtcgt
     1381 aaacggatgc tggctagtgc cggagaatta caaaaaggaa atgagctggc tctgccaagc
     1441 aaatatgtga attttttata tttagctagt cattatgaaa agttgaaggg tagtccagaa
     1501 gataacgaac aaaaacaatt gtttgtggag cagcataagc attatttaga tgagattatt
     1561 gagcaaatca gtgaattttc taagcgtgtt attttagcag atgccaattt agataaagtt
     1621 cttagtgcat ataacaaaca tagagacaaa ccaatacgtg aacaagcaga aaatattatt
     1681 catttattta cgttgacgaa tcttggagct cccgctgctt ttaaatattt tgatacaaca
     1741 attgatcgta aacgatatac gtctacaaaa gaagttttag atgccactct tatccatcaa
     1801 tccatcactg gtctttatga aacacgcatt gatttgagtc agctaggagg tgacggcgga
     1861 tcaggaggct ctggcggttc aggtggatca ggcggtagcg gaggttcagg tggtatggat
     1921 aagaaatact caataggctt agctatcggc acaaatagcg tcggatgggc ggtgatcact
     1981 gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga ccgccacagt
     2041 atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc ggaagcgact
     2101 cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat ttgttatcta
     2161 caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca tcgacttgaa
     2221 gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt tggaaatata
     2281 gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg aaaaaaattg
     2341 gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt
     2401 aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac
     2461 aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac
     2521 gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta
     2581 gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg gaatctcatt
     2641 gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc agaagatgct
     2701 aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt ggcgcaaatt
     2761 ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt
     2821 tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt
     2881 aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa
     2941 cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat
     3001 attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa
     3061 atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa
     3121 cggacctttg acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt
     3181 ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa
     3241 atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt
     3301 gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc
     3361 gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga taaaaatctt
     3421 ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac
     3481 gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt
     3541 gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag
     3601 caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga
     3661 gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa
     3721 gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca
     3781 ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc
     3841 tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggacgtttg
     3901 tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt
     3961 ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga tgatagtttg
     4021 acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa
     4081 catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa
     4141 gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa
     4201 atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa
     4261 cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa
     4321 aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg
     4381 tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga tgccattgtt
     4441 ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa
     4501 aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat
     4561 tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa
     4621 gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt
     4681 gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa
     4741 tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta
     4801 gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat
     4861 catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca
     4921 aaacttgaat cggagtttgt ctatggttga 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
//
