LOCUS       sequence-231655-        6881 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TetR_tetR/tetA promoters_bCas9-CP199-TEVlinker-2xflag_CM_P15A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6881)
  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 6881)
  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 36.02_ProCas9TEV
FEATURES             Location/Qualifiers
     source          1..6881
                     /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"
     CDS             4288..4308
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4918..4965
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     terminator      5046..5117
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5133..5160
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5188..5205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5322..5867)
                     /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(5402..5421)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5981..6075)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6096..6755)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6670..6689
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6756..6858)
                     /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 aggtgacgaa aatctttatt ttcaatccat ggataagaag
     4321 tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
     4381 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
     4441 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
     4501 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
     4561 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
     4621 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
     4681 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
     4741 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
     4801 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
     4861 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctcc cgggggtgat
     4921 tacaaggatg atgatgataa ggattacaag gatgatgatg ataagtgatt gattgaacga
     4981 aaaacgcgaa agcgtttcac gataaatgcg aaaacggatc ctctcgagta aggatctcca
     5041 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     5101 tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg
     5161 tttataccta gggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg
     5221 actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa
     5281 gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc
     5341 cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta
     5401 taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt tcctgccttt
     5461 cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac gcctgacact
     5521 cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc ccgttcagtc
     5581 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa
     5641 agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg aagtcatgcg
     5701 ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca agccagttac
     5761 ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc aaggcggttt
     5821 tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa gatcatctta
     5881 ttaatcagat aaaatatttc tagatttcag tgcaatttat ctcttcaaat gtagcacctg
     5941 aagtcagccc catacgatat aagttgttac tagtgcttgg attctcacca ataaaaaacg
     6001 cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat
     6061 ctatcaacag gagtccaagc gagctcgata tcaaattacg ccccgccctg ccactcatcg
     6121 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
     6181 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
     6241 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
     6301 ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag
     6361 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
     6421 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
     6481 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacgaaat
     6541 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
     6601 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
     6661 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
     6721 tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
     6781 ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa
     6841 cctcttacgt gccgatcaac gtctcatttt cgccagatat c
//
