LOCUS       sequence-231304-        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 9.3_bCas9-CP230
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 ttatatgacc
      781 ggtgagaaga aaaatggctt atttgggaat ctcattgctt tgtcattggg tttgacccct
      841 aattttaaat caaattttga tttggcagaa gatgctaaat tacagctttc aaaagatact
      901 tacgatgatg atttagataa tttattggcg caaattggag atcaatatgc tgatttgttt
      961 ttggcagcta agaatttatc agatgctatt ttactttcag atatcctaag agtaaatact
     1021 gaaataacta aggctcccct atcagcttca atgattaaac gctacgatga acatcatcaa
     1081 gacttgactc ttttaaaagc tttagttcga caacaacttc cagaaaagta taaagaaatc
     1141 ttttttgatc aatcaaaaaa cggatatgca ggttatattg atgggggagc tagccaagaa
     1201 gaattttata aatttatcaa accaatttta gaaaaaatgg atggtactga ggaattattg
     1261 gtgaaactaa atcgtgaaga tttgctgcgc aagcaacgga cctttgacaa cggctctatt
     1321 ccccatcaaa ttcacttggg tgagctgcat gctattttga gaagacaaga agacttttat
     1381 ccatttttaa aagacaatcg tgagaagatt gaaaaaatct tgacttttcg aattccttat
     1441 tatgttggtc cattggcgcg tggcaatagt cgttttgcat ggatgactcg gaagtctgaa
     1501 gaaacaatta ccccatggaa ttttgaagaa gttgtcgata aaggtgcttc agctcaatca
     1561 tttattgaac gcatgacaaa ctttgataaa aatcttccaa atgaaaaagt actaccaaaa
     1621 catagtttgc tttatgagta ttttacggtt tataacgaat tgacaaaggt caaatatgtt
     1681 actgaaggaa tgcgaaaacc agcatttctt tcaggtgaac agaagaaagc cattgttgat
     1741 ttactcttca aaacaaatcg aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa
     1801 aaaatagaat gttttgatag tgttgaaatt tcaggagttg aagatagatt taatgcttca
     1861 ttaggtacct accatgattt gctaaaaatt attaaagata aagatttttt ggataatgaa
     1921 gaaaatgaag atatcttaga ggatattgtt ttaacattga ccttatttga agatagggag
     1981 atgattgagg aaagacttaa aacatatgct cacctctttg atgataaggt gatgaaacag
     2041 cttaaacgtc gccgttatac tggttgggga cgtttgtctc gaaaattgat taatggtatt
     2101 agggataagc aatctggcaa aacaatatta gattttttga aatcagatgg ttttgccaat
     2161 cgcaatttta tgcagctgat ccatgatgat agtttgacat ttaaagaaga cattcaaaaa
     2221 gcacaagtgt ctggacaagg cgatagttta catgaacata ttgcaaattt agctggtagc
     2281 cctgctatta aaaaaggtat tttacagact gtaaaagttg ttgatgaatt ggtcaaagta
     2341 atggggcggc ataagccaga aaatatcgtt attgaaatgg cacgtgaaaa tcagacaact
     2401 caaaagggcc agaaaaattc gcgagagcgt atgaaacgaa tcgaagaagg tatcaaagaa
     2461 ttaggaagtc agattcttaa agagcatcct gttgaaaata ctcaattgca aaatgaaaag
     2521 ctctatctct attatctcca aaatggaaga gacatgtatg tggaccaaga attagatatt
     2581 aatcgtttaa gtgattatga tgtcgatgcc attgttccac aaagtttcct taaagacgat
     2641 tcaatagaca ataaggtctt aacgcgttct gataaaaatc gtggtaaatc ggataacgtt
     2701 ccaagtgaag aagtagtcaa aaagatgaaa aactattgga gacaacttct aaacgccaag
     2761 ttaatcactc aacgtaagtt tgataattta acgaaagctg aacgtggagg tttgagtgaa
     2821 cttgataaag ctggttttat caaacgccaa ttggttgaaa ctcgccaaat cactaagcat
     2881 gtggcacaaa ttttggatag tcgcatgaat actaaatacg atgaaaatga taaacttatt
     2941 cgagaggtta aagtgattac cttaaaatct aaattagttt ctgacttccg aaaagatttc
     3001 caattctata aagtacgtga gattaacaat taccatcatg cccatgatgc gtatctaaat
     3061 gccgtcgttg gaactgcttt gattaagaaa tatccaaaac ttgaatcgga gtttgtctat
     3121 ggtgattata aagtttatga tgttcgtaaa atgattgcta agtctgagca agaaataggc
     3181 aaagcaaccg caaaatattt cttttactct aatatcatga acttcttcaa aacagaaatt
     3241 acacttgcaa atggagagat tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga
     3301 gaaattgtct gggataaagg gcgagatttt gccacagtgc gcaaagtatt gtccatgccc
     3361 caagtcaata ttgtcaagaa aacagaagta cagacaggcg gattctccaa ggagtcaatt
     3421 ttaccaaaaa gaaattcgga caagcttatt gctcgtaaaa aagactggga tccaaaaaaa
     3481 tatggtggtt ttgatagtcc aacggtagct tattcagtcc tagtggttgc taaggtggaa
     3541 aaagggaaat cgaagaagtt aaaatccgtt aaagagttac tagggatcac aattatggaa
     3601 agaagttcct ttgaaaaaaa tccgattgac tttttagaag ctaaaggata taaggaagtt
     3661 aaaaaagact taatcattaa actacctaaa tatagtcttt ttgagttaga aaacggtcgt
     3721 aaacggatgc tggctagtgc cggagaatta caaaaaggaa atgagctggc tctgccaagc
     3781 aaatatgtga attttttata tttagctagt cattatgaaa agttgaaggg tagtccagaa
     3841 gataacgaac aaaaacaatt gtttgtggag cagcataagc attatttaga tgagattatt
     3901 gagcaaatca gtgaattttc taagcgtgtt attttagcag atgccaattt agataaagtt
     3961 cttagtgcat ataacaaaca tagagacaaa ccaatacgtg aacaagcaga aaatattatt
     4021 catttattta cgttgacgaa tcttggagct cccgctgctt ttaaatattt tgatacaaca
     4081 attgatcgta aacgatatac gtctacaaaa gaagttttag atgccactct tatccatcaa
     4141 tccatcactg gtctttatga aacacgcatt gatttgagtc agctaggagg tgacggcgga
     4201 tcaggaggct ctggcggttc aggtggatca ggcggtagcg gaggttcagg tggtatggat
     4261 aagaaatact caataggctt agctatcggc acaaatagcg tcggatgggc ggtgatcact
     4321 gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga ccgccacagt
     4381 atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc ggaagcgact
     4441 cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat ttgttatcta
     4501 caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca tcgacttgaa
     4561 gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt tggaaatata
     4621 gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg aaaaaaattg
     4681 gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt
     4741 aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac
     4801 aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac
     4861 gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta
     4921 gaaaatctca ttgctcagct ccccggttga 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
//
