LOCUS       sequence-237525-        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 9.2_bCas9-CP199
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 ttatatgaac
      781 cctattaacg caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca
      841 agacgattag aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg
      901 aatctcattg ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca
      961 gaagatgcta aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg
     1021 gcgcaaattg gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct
     1081 attttacttt cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct
     1141 tcaatgatta aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt
     1201 cgacaacaac ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat
     1261 gcaggttata ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt
     1321 ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg
     1381 cgcaagcaac ggacctttga caacggctct attccccatc aaattcactt gggtgagctg
     1441 catgctattt tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag
     1501 attgaaaaaa tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat
     1561 agtcgttttg catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa
     1621 gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat
     1681 aaaaatcttc caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg
     1741 gtttataacg aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt
     1801 ctttcaggtg aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta
     1861 accgttaagc aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa
     1921 atttcaggag ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa
     1981 attattaaag ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt
     2041 gttttaacat tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat
     2101 gctcacctct ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg
     2161 ggacgtttgt ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata
     2221 ttagattttt tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat
     2281 gatagtttga catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt
     2341 ttacatgaac atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag
     2401 actgtaaaag ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc
     2461 gttattgaaa tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag
     2521 cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat
     2581 cctgttgaaa atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga
     2641 agagacatgt atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat
     2701 gccattgttc cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt
     2761 tctgataaaa atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg
     2821 aaaaactatt ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat
     2881 ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc
     2941 caattggttg aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg
     3001 aatactaaat acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa
     3061 tctaaattag tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac
     3121 aattaccatc atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag
     3181 aaatatccaa aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt
     3241 aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac
     3301 tctaatatca tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa
     3361 cgccctctaa tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat
     3421 tttgccacag tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa
     3481 gtacagacag gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt
     3541 attgctcgta aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta
     3601 gcttattcag tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc
     3661 gttaaagagt tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt
     3721 gactttttag aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct
     3781 aaatatagtc tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa
     3841 ttacaaaaag gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct
     3901 agtcattatg aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg
     3961 gagcagcata agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt
     4021 gttattttag cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac
     4081 aaaccaatac gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga
     4141 gctcccgctg cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca
     4201 aaagaagttt tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc
     4261 attgatttga gtcagctagg aggtgacggc ggatcaggag gctctggcgg ttcaggtgga
     4321 tcaggcggta gcggaggttc aggtggtatg gataagaaat actcaatagg cttagctatc
     4381 ggcacaaata gcgtcggatg ggcggtgatc actgatgaat ataaggttcc gtctaaaaag
     4441 ttcaaggttc tgggaaatac agaccgccac agtatcaaaa aaaatcttat aggggctctt
     4501 ttatttgaca gtggagagac agcggaagcg actcgtctca aacggacagc tcgtagaagg
     4561 tatacacgtc ggaagaatcg tatttgttat ctacaggaga ttttttcaaa tgagatggcg
     4621 aaagtagatg atagtttctt tcatcgactt gaagagtctt ttttggtgga agaagacaag
     4681 aagcatgaac gtcatcctat ttttggaaat atagtagatg aagttgctta tcatgagaaa
     4741 tatccaacta tctatcatct gcgaaaaaaa ttggtagatt ctactgataa agcggatttg
     4801 cgcttaatct atttggcctt agcgcatatg attaagtttc gtggtcattt tttgattgag
     4861 ggagatttaa atcctgataa tagtgatgtg gacaaactat ttatccagtt ggtacaaacc
     4921 tacaatcaat tatttgaaga aaacccttga 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
//
