LOCUS       sequence-237537-        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 9.15_bCas9-CP1249
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 ttatatgaca
      781 gaagataacg aacaaaaaca attgtttgtg gagcagcata agcattattt agatgagatt
      841 attgagcaaa tcagtgaatt ttctaagcgt gttattttag cagatgccaa tttagataaa
      901 gttcttagtg catataacaa acatagagac aaaccaatac gtgaacaagc agaaaatatt
      961 attcatttat ttacgttgac gaatcttgga gctcccgctg cttttaaata ttttgataca
     1021 acaattgatc gtaaacgata tacgtctaca aaagaagttt tagatgccac tcttatccat
     1081 caatccatca ctggtcttta tgaaacacgc attgatttga gtcagctagg aggtgacggc
     1141 ggatcaggag gctctggcgg ttcaggtgga tcaggcggta gcggaggttc aggtggtatg
     1201 gataagaaat actcaatagg cttagctatc ggcacaaata gcgtcggatg ggcggtgatc
     1261 actgatgaat ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac agaccgccac
     1321 agtatcaaaa aaaatcttat aggggctctt ttatttgaca gtggagagac agcggaagcg
     1381 actcgtctca aacggacagc tcgtagaagg tatacacgtc ggaagaatcg tatttgttat
     1441 ctacaggaga ttttttcaaa tgagatggcg aaagtagatg atagtttctt tcatcgactt
     1501 gaagagtctt ttttggtgga agaagacaag aagcatgaac gtcatcctat ttttggaaat
     1561 atagtagatg aagttgctta tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa
     1621 ttggtagatt ctactgataa agcggatttg cgcttaatct atttggcctt agcgcatatg
     1681 attaagtttc gtggtcattt tttgattgag ggagatttaa atcctgataa tagtgatgtg
     1741 gacaaactat ttatccagtt ggtacaaacc tacaatcaat tatttgaaga aaaccctatt
     1801 aacgcaagtg gagtagatgc taaagcgatt ctttctgcac gattgagtaa atcaagacga
     1861 ttagaaaatc tcattgctca gctccccggt gagaagaaaa atggcttatt tgggaatctc
     1921 attgctttgt cattgggttt gacccctaat tttaaatcaa attttgattt ggcagaagat
     1981 gctaaattac agctttcaaa agatacttac gatgatgatt tagataattt attggcgcaa
     2041 attggagatc aatatgctga tttgtttttg gcagctaaga atttatcaga tgctatttta
     2101 ctttcagata tcctaagagt aaatactgaa ataactaagg ctcccctatc agcttcaatg
     2161 attaaacgct acgatgaaca tcatcaagac ttgactcttt taaaagcttt agttcgacaa
     2221 caacttccag aaaagtataa agaaatcttt tttgatcaat caaaaaacgg atatgcaggt
     2281 tatattgatg ggggagctag ccaagaagaa ttttataaat ttatcaaacc aattttagaa
     2341 aaaatggatg gtactgagga attattggtg aaactaaatc gtgaagattt gctgcgcaag
     2401 caacggacct ttgacaacgg ctctattccc catcaaattc acttgggtga gctgcatgct
     2461 attttgagaa gacaagaaga cttttatcca tttttaaaag acaatcgtga gaagattgaa
     2521 aaaatcttga cttttcgaat tccttattat gttggtccat tggcgcgtgg caatagtcgt
     2581 tttgcatgga tgactcggaa gtctgaagaa acaattaccc catggaattt tgaagaagtt
     2641 gtcgataaag gtgcttcagc tcaatcattt attgaacgca tgacaaactt tgataaaaat
     2701 cttccaaatg aaaaagtact accaaaacat agtttgcttt atgagtattt tacggtttat
     2761 aacgaattga caaaggtcaa atatgttact gaaggaatgc gaaaaccagc atttctttca
     2821 ggtgaacaga agaaagccat tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt
     2881 aagcaattaa aagaagatta tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca
     2941 ggagttgaag atagatttaa tgcttcatta ggtacctacc atgatttgct aaaaattatt
     3001 aaagataaag attttttgga taatgaagaa aatgaagata tcttagagga tattgtttta
     3061 acattgacct tatttgaaga tagggagatg attgaggaaa gacttaaaac atatgctcac
     3121 ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc gttatactgg ttggggacgt
     3181 ttgtctcgaa aattgattaa tggtattagg gataagcaat ctggcaaaac aatattagat
     3241 tttttgaaat cagatggttt tgccaatcgc aattttatgc agctgatcca tgatgatagt
     3301 ttgacattta aagaagacat tcaaaaagca caagtgtctg gacaaggcga tagtttacat
     3361 gaacatattg caaatttagc tggtagccct gctattaaaa aaggtatttt acagactgta
     3421 aaagttgttg atgaattggt caaagtaatg gggcggcata agccagaaaa tatcgttatt
     3481 gaaatggcac gtgaaaatca gacaactcaa aagggccaga aaaattcgcg agagcgtatg
     3541 aaacgaatcg aagaaggtat caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt
     3601 gaaaatactc aattgcaaaa tgaaaagctc tatctctatt atctccaaaa tggaagagac
     3661 atgtatgtgg accaagaatt agatattaat cgtttaagtg attatgatgt cgatgccatt
     3721 gttccacaaa gtttccttaa agacgattca atagacaata aggtcttaac gcgttctgat
     3781 aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac
     3841 tattggagac aacttctaaa cgccaagtta atcactcaac gtaagtttga taatttaacg
     3901 aaagctgaac gtggaggttt gagtgaactt gataaagctg gttttatcaa acgccaattg
     3961 gttgaaactc gccaaatcac taagcatgtg gcacaaattt tggatagtcg catgaatact
     4021 aaatacgatg aaaatgataa acttattcga gaggttaaag tgattacctt aaaatctaaa
     4081 ttagtttctg acttccgaaa agatttccaa ttctataaag tacgtgagat taacaattac
     4141 catcatgccc atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat taagaaatat
     4201 ccaaaacttg aatcggagtt tgtctatggt gattataaag tttatgatgt tcgtaaaatg
     4261 attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt ttactctaat
     4321 atcatgaact tcttcaaaac agaaattaca cttgcaaatg gagagattcg caaacgccct
     4381 ctaatcgaaa ctaatgggga aactggagaa attgtctggg ataaagggcg agattttgcc
     4441 acagtgcgca aagtattgtc catgccccaa gtcaatattg tcaagaaaac agaagtacag
     4501 acaggcggat tctccaagga gtcaatttta ccaaaaagaa attcggacaa gcttattgct
     4561 cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg atagtccaac ggtagcttat
     4621 tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa
     4681 gagttactag ggatcacaat tatggaaaga agttcctttg aaaaaaatcc gattgacttt
     4741 ttagaagcta aaggatataa ggaagttaaa aaagacttaa tcattaaact acctaaatat
     4801 agtctttttg agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa
     4861 aaaggaaatg agctggctct gccaagcaaa tatgtgaatt ttttatattt agctagtcat
     4921 tatgaaaagt tgaagggtag tccagaatga 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
//
