LOCUS       sequence-246183-        9132 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  derivative of pFD116 with an optimized RBS for E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9132)
  AUTHORS   Depardieu F, Bikard D
  TITLE     Gene silencing with CRISPRi in bacteria and optimization of dCas9 
            expression levels.
  JOURNAL   Methods. 2019 Aug 1. pii: S1046-2023(18)30497-3. doi: 
            10.1016/j.ymeth.2019.07.024.
  PUBMED    31377338
REFERENCE   2  (bases 1 to 9132)
  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: "Methods. 
            2019 Aug 1. pii: S1046-2023(18)30497-3. doi: 
            10.1016/j.ymeth.2019.07.024."
COMMENT     Sequence Label: pFD152
FEATURES             Location/Qualifiers
     source          1..9132
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      56..127
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      143..170
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        381..456
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     oriT            631..740
                     /note="incP origin of transfer"
     CDS             complement(1616..1627)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(4254..4880)
                     /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     4785..4804
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             join(5073..5099,5100..5102,5103..7589,7590..7592,7593..9132,
                     1..47)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gtctttatga aacacgcatt gatttgagtc agctaggagg tgactgacca ggcatcaaat
       61 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
      121 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataatat
      181 actcctaaat taacttttaa agcaatgaaa atagtgaaca ttataactgt tgtgtaacag
      241 aatgcaatta gcatattact gttacacaaa ttagtacagt ttctatgttt tgacatacat
      301 ttgatgaaaa ttgtacataa tttatgtgaa aaaaatcaca acaaacatgc tacaattagt
      361 tgagaccagt ctaggtctcg gttttagagc tagaaatagc aagttaaaat aaggctagtc
      421 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttggagatc tgtccatacc
      481 catggattct tcgtctgttt ctactccatc agtctgacga ccaagagagc cataaacacc
      541 aatagcctta acatcatccc catatttatc caatattcgt tccttaattt catgaacaat
      601 ctgatatcaa gcttatcgat accgtcgacg ccggccagcc tcgcagagca ggattcccgt
      661 tgagcaccgc caggtgcgaa taagggacag tgaagaagga acacccgctc gcgggtgggc
      721 ctacttcacc tatcctgccc ggctgacgcc gttggataca ccaaggaaag tctacacgaa
      781 ccctttggca aaatcctgta tatcgtgcga aaaaggatgg atataccgaa aaaatcgcta
      841 taatgacccc gaagcagggt tatgcagcgg aaaagcgctg gtacccaatt cgccctatag
      901 tgagtcttct tctctagtca ttattattgg tccattcact attctcattc ccttttcaga
      961 taattttaga tttgcttttc taaataagaa tatttggaga gcaccgttct tattcagcta
     1021 ttaataactc gtcttcctaa gcatccttca atccttttaa taacaattat agcatctaat
     1081 cttcaacaaa ctggcccgtt tgttgaacta ctctttaata aaataatttt tccgttccca
     1141 attccacatt gcaataatag aaaatccatc ttcatcggct ttttcgtcat catctgtatg
     1201 aatcaaatcg ccttcttctg tgtcatcaag gtttaatttt ttatgtattt cttttaacaa
     1261 accaccatag gagattaacc ttttacggtg taaaccttcc tccaaatcag acaaacgttt
     1321 caaattcttt tcttcatcat cggtcataaa atccgtatcc tttacaggat attttgcagt
     1381 ttcgtcaatt gccgattgta tatccgattt atatttattt ttcggtcgat tttttattaa
     1441 aacgtctcaa aatcgtttct gagacgtttt agcgtttatt tcgtttagtt atcggcataa
     1501 tcgttaaaac aggcgttatc gtagcgtaaa agcccttgag cgtagcgtgg ctttgcagcg
     1561 aagatgttgt ctgttagatt atgaaagccg atgactgaat gaaataataa gcgcagcgtc
     1621 cttctatttc ggttggagga ggctcaaggg agtttgaggg aatgaaattc cctcatgggt
     1681 ttgattttaa aaattgcttg caattttgcc gagcggtagc gctggaaaat ttttgaaaaa
     1741 aatttggaat ttggaaaaaa atggggggaa aggaagcgaa ttttgcttcc gtactacgac
     1801 ccccccatta agtgccgagt gccaattttt gtgccaaaaa cgctctatcc caactggctc
     1861 aagggtttga ggggtttttc aatcgccaac gaatcgccaa cgttttcgcc aacgtttttt
     1921 ataaatctat atttaagtag ctttattgtt gtttttatga ttacaaagtg atacactaat
     1981 tttataaaat tatttgattg gagtttttta aatggtgatt tcagaatcga aaaaaagagt
     2041 tatgatttct ctgacaaaag agcaagataa aaaattaaca gatatggcga aacaaaaagg
     2101 tttttcaaaa tctgcggttg cggcgttagc tatagaagaa tatgcaagaa aggaatcaga
     2161 acaaaaaaaa taagcgaaag ctcgcgtttt tagaaggata cgagttttcg ctacttgttt
     2221 ttgataaggt aattatatca tggctattaa aaatactaaa gctagaaatt ttggattttt
     2281 attatatcct gactcaattc ctaatgattg gaaagaaaaa ttagagagtt tgggcgtatc
     2341 tatggctgtc agtcctttac acgatatgga cgaaaaaaaa gataaagata catggaatag
     2401 tagtgatgtt atacgaaatg gaaagcacta taaaaaacca cactatcacg ttatatatat
     2461 tgcacgaaat cctgtaacaa tagaaagcgt taggaacaag attaagcgaa aattggggaa
     2521 tagttcagtt gctcatgttg agatacttga ttatatcaaa ggttcatatg aatatttgac
     2581 tcatgaatca aaggacgcta ttgctaagaa taaacatata tacgacaaaa aagatatttt
     2641 gaacattaat gattttgata ttgaccgcta tataacactt gatgaaagcc aaaaaagaaa
     2701 attgaagaat ttacttttag atatagtgga tgactataat ttggtaaata caaaagattt
     2761 aatggctttt attcgcctta ggggagcgga gtttggaatt ttaaatacga atgatgtaaa
     2821 agatattgtt tcaacaaact ctagcgcctt tagattatgg tttgagggca attatcagtg
     2881 tggatataga gcaagttatg caaaggttct tgatgctgaa acgggggaaa taaaatgaca
     2941 aacaaagaaa aagagttatt tgctgaaaat gaggaattaa aaaaagaaat taaggactta
     3001 aaagagcgta ttgaaagata cagagaaatg gaagttgaat taagtacaac aatagattta
     3061 ttgagaggag ggattattga ataaataaaa gcccccctga cgaaagtcca agggtttatt
     3121 gttttctaaa atctgattac caattagaat gaatatttcc caaatattaa ataataaaac
     3181 aaaaaaatta aaaaagtgtt tccaccattt tttcaatttt tttataattt ttttaatctg
     3241 ttatttaaat agtttatagt taaatttaca ttttcattag tccattcaat attctctcca
     3301 agataactac gaactgctaa caaaattctc tccctatgtt ctaatggaga agattcagcc
     3361 actgcatttc ccgcaatatc ttttggtatg attttacccg tgtccatagt taaaatcata
     3421 cggcataaag ttaatataga gttggtttca tcatcctgat aattatctat taattcctct
     3481 gacgaatcca taatggctct tctcacatca gaaaatggaa tatcaggtag taattcctct
     3541 aagtcataat ttccgtatat tcttttattt tttcgttttg cttggtaaag cattatggtt
     3601 aaatctgaat ttaattcctt ctgaggaatg tatccttgtt cataaagctc ttgtaaccat
     3661 tctccataaa taaattcttg tttgggagga tgattccacg gtaccatttc ttgctgaata
     3721 ataattgtta attcaatata tcgtaagttg cttttatctc ctattttttt tgaaataggt
     3781 ctaatttttt gtataagtat ttctttactt tgatctgtca atggttcaga tacgacgact
     3841 aaaaagtcaa gatcactatt tggttttagt ccactctcaa ctcctgatcc aaacatgtaa
     3901 gtaccaataa ggttattttt taaatgtttc cgaagtattt ttttcacttt attaatttgt
     3961 tcgtatgtat tcaaatatat cctcctcact attttgatta gtacctattt tatatccata
     4021 gttgttaatt aaataaactt aatttagttt atttatagat ttcattggct tctaaatttt
     4081 ttatctagat aataattatt ttagttaatt ttattctaga ttatatatga tatgatcttt
     4141 catttccata aaactaaagt aagtgtaaac ctattcattg ttttaaaaat atctcttgcc
     4201 agtcacgtta cgttattagt tagttaccat cacggaaaaa ggttatgctg cttttaagac
     4261 ccactttcac atttaagttg tttttctaat ccgcatatga tcaattcaag gccgaataag
     4321 aaggctggct ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa taatggcggc
     4381 atactatcag tagtaggtgt ttccctttct tctttagcga cttgatgctc ttgatcttcc
     4441 aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg catataatgc attctctagt
     4501 gaaaaacctt gttggcataa aaaggctaat tgattttcga gagtttcata ctgtttttct
     4561 gtaggccgtg tacctaaatg tacttttgct ccatcgcgat gacttagtaa agcacatcta
     4621 aaacttttag cgttattacg taaaaaatct tgccagcttt ccccttctaa agggcaaaag
     4681 tgagtatggt gcctatctaa catctcaatg gctaaggcgt cgagcaaagc ccgcttattt
     4741 tttacatgcc aatacaatgt aggctgctct acacctagct tctgggcgag tttacgggtt
     4801 gttaaacctt cgattccgac ctcattaagc agctctaatg cgctgttaat cactttactt
     4861 ttatctaatc tagacatcat taattcctcc tttttgttga cattatatca ttgatagagt
     4921 tatttgtcaa actagttttt tatttggatc ccctcgagtt catgaaaaac taaaaaaaat
     4981 attgacactc tatcattgat agagcataat taaaataagc tctctatcat tgatagagtg
     5041 atatcgaatt cggacgcata gtggcctcaa aaatggataa gaaatactca ataggcttag
     5101 ctatcggcac aaatagcgtc ggatgggcgg tgatcactga tgaatataag gttccgtcta
     5161 aaaagttcaa ggttctggga aatacagacc gccacagtat caaaaaaaat cttatagggg
     5221 ctcttttatt tgacagtgga gagacagcgg aagcgactcg tctcaaacgg acagctcgta
     5281 gaaggtatac acgtcggaag aatcgtattt gttatctaca ggagattttt tcaaatgaga
     5341 tggcgaaagt agatgatagt ttctttcatc gacttgaaga gtcttttttg gtggaagaag
     5401 acaagaagca tgaacgtcat cctatttttg gaaatatagt agatgaagtt gcttatcatg
     5461 agaaatatcc aactatctat catctgcgaa aaaaattggt agattctact gataaagcgg
     5521 atttgcgctt aatctatttg gccttagcgc atatgattaa gtttcgtggt cattttttga
     5581 ttgagggaga tttaaatcct gataatagtg atgtggacaa actatttatc cagttggtac
     5641 aaacctacaa tcaattattt gaagaaaacc ctattaacgc aagtggagta gatgctaaag
     5701 cgattctttc tgcacgattg agtaaatcaa gacgattaga aaatctcatt gctcagctcc
     5761 ccggtgagaa gaaaaatggc ttatttggga atctcattgc tttgtcattg ggtttgaccc
     5821 ctaattttaa atcaaatttt gatttggcag aagatgctaa attacagctt tcaaaagata
     5881 cttacgatga tgatttagat aatttattgg cgcaaattgg agatcaatat gctgatttgt
     5941 ttttggcagc taagaattta tcagatgcta ttttactttc agatatccta agagtaaata
     6001 ctgaaataac taaggctccc ctatcagctt caatgattaa acgctacgat gaacatcatc
     6061 aagacttgac tcttttaaaa gctttagttc gacaacaact tccagaaaag tataaagaaa
     6121 tcttttttga tcaatcaaaa aacggatatg caggttatat tgatggggga gctagccaag
     6181 aagaatttta taaatttatc aaaccaattt tagaaaaaat ggatggtact gaggaattat
     6241 tggtgaaact aaatcgtgaa gatttgctgc gcaagcaacg gacctttgac aacggctcta
     6301 ttccccatca aattcacttg ggtgagctgc atgctatttt gagaagacaa gaagactttt
     6361 atccattttt aaaagacaat cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt
     6421 attatgttgg tccattggcg cgtggcaata gtcgttttgc atggatgact cggaagtctg
     6481 aagaaacaat taccccatgg aattttgaag aagttgtcga taaaggtgct tcagctcaat
     6541 catttattga acgcatgaca aactttgata aaaatcttcc aaatgaaaaa gtactaccaa
     6601 aacatagttt gctttatgag tattttacgg tttataacga attgacaaag gtcaaatatg
     6661 ttactgaagg aatgcgaaaa ccagcatttc tttcaggtga acagaagaaa gccattgttg
     6721 atttactctt caaaacaaat cgaaaagtaa ccgttaagca attaaaagaa gattatttca
     6781 aaaaaataga atgttttgat agtgttgaaa tttcaggagt tgaagataga tttaatgctt
     6841 cattaggtac ctaccatgat ttgctaaaaa ttattaaaga taaagatttt ttggataatg
     6901 aagaaaatga agatatctta gaggatattg ttttaacatt gaccttattt gaagataggg
     6961 agatgattga ggaaagactt aaaacatatg ctcacctctt tgatgataag gtgatgaaac
     7021 agcttaaacg tcgccgttat actggttggg gacgtttgtc tcgaaaattg attaatggta
     7081 ttagggataa gcaatctggc aaaacaatat tagatttttt gaaatcagat ggttttgcca
     7141 atcgcaattt tatgcagctg atccatgatg atagtttgac atttaaagaa gacattcaaa
     7201 aagcacaagt gtctggacaa ggcgatagtt tacatgaaca tattgcaaat ttagctggta
     7261 gccctgctat taaaaaaggt attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag
     7321 taatggggcg gcataagcca gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa
     7381 ctcaaaaggg ccagaaaaat tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag
     7441 aattaggaag tcagattctt aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa
     7501 agctctatct ctattatctc caaaatggaa gagacatgta tgtggaccaa gaattagata
     7561 ttaatcgttt aagtgattat gatgtcgatg ccattgttcc acaaagtttc cttaaagacg
     7621 attcaataga caataaggtc ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg
     7681 ttccaagtga agaagtagtc aaaaagatga aaaactattg gagacaactt ctaaacgcca
     7741 agttaatcac tcaacgtaag tttgataatt taacgaaagc tgaacgtgga ggtttgagtg
     7801 aacttgataa agctggtttt atcaaacgcc aattggttga aactcgccaa atcactaagc
     7861 atgtggcaca aattttggat agtcgcatga atactaaata cgatgaaaat gataaactta
     7921 ttcgagaggt taaagtgatt accttaaaat ctaaattagt ttctgacttc cgaaaagatt
     7981 tccaattcta taaagtacgt gagattaaca attaccatca tgcccatgat gcgtatctaa
     8041 atgccgtcgt tggaactgct ttgattaaga aatatccaaa acttgaatcg gagtttgtct
     8101 atggtgatta taaagtttat gatgttcgta aaatgattgc taagtctgag caagaaatag
     8161 gcaaagcaac cgcaaaatat ttcttttact ctaatatcat gaacttcttc aaaacagaaa
     8221 ttacacttgc aaatggagag attcgcaaac gccctctaat cgaaactaat ggggaaactg
     8281 gagaaattgt ctgggataaa gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc
     8341 cccaagtcaa tattgtcaag aaaacagaag tacagacagg cggattctcc aaggagtcaa
     8401 ttttaccaaa aagaaattcg gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa
     8461 aatatggtgg ttttgatagt ccaacggtag cttattcagt cctagtggtt gctaaggtgg
     8521 aaaaagggaa atcgaagaag ttaaaatccg ttaaagagtt actagggatc acaattatgg
     8581 aaagaagttc ctttgaaaaa aatccgattg actttttaga agctaaagga tataaggaag
     8641 ttaaaaaaga cttaatcatt aaactaccta aatatagtct ttttgagtta gaaaacggtc
     8701 gtaaacggat gctggctagt gccggagaat tacaaaaagg aaatgagctg gctctgccaa
     8761 gcaaatatgt gaatttttta tatttagcta gtcattatga aaagttgaag ggtagtccag
     8821 aagataacga acaaaaacaa ttgtttgtgg agcagcataa gcattattta gatgagatta
     8881 ttgagcaaat cagtgaattt tctaagcgtg ttattttagc agatgccaat ttagataaag
     8941 ttcttagtgc atataacaaa catagagaca aaccaatacg tgaacaagca gaaaatatta
     9001 ttcatttatt tacgttgacg aatcttggag ctcccgctgc ttttaaatat tttgatacaa
     9061 caattgatcg taaacgatat acgtctacaa aagaagtttt agatgccact cttatccatc
     9121 aatccatcac tg
//
