LOCUS       sequence-499185-        9131 bp    DNA     circular SYN 05-FEB-2026
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 9131)
  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 9131)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 5, 2026 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..9131
                     /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(4253..4879)
                     /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     4784..4803
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             join(5072..5098,5099..5101,5102..7588,7589..7591,7592..9131,
                     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 cccccattaa gtgccgagtg ccaatttttg tgccaaaaac gctctatccc aactggctca
     1861 agggtttgag gggtttttca atcgccaacg aatcgccaac gttttcgcca acgtttttta
     1921 taaatctata tttaagtagc tttattgttg tttttatgat tacaaagtga tacactaatt
     1981 ttataaaatt atttgattgg agttttttaa atggtgattt cagaatcgaa aaaaagagtt
     2041 atgatttctc tgacaaaaga gcaagataaa aaattaacag atatggcgaa acaaaaaggt
     2101 ttttcaaaat ctgcggttgc ggcgttagct atagaagaat atgcaagaaa ggaatcagaa
     2161 caaaaaaaat aagcgaaagc tcgcgttttt agaaggatac gagttttcgc tacttgtttt
     2221 tgataaggta attatatcat ggctattaaa aatactaaag ctagaaattt tggattttta
     2281 ttatatcctg actcaattcc taatgattgg aaagaaaaat tagagagttt gggcgtatct
     2341 atggctgtca gtcctttaca cgatatggac gaaaaaaaag ataaagatac atggaatagt
     2401 agtgatgtta tacgaaatgg aaagcactat aaaaaaccac actatcacgt tatatatatt
     2461 gcacgaaatc ctgtaacaat agaaagcgtt aggaacaaga ttaagcgaaa attggggaat
     2521 agttcagttg ctcatgttga gatacttgat tatatcaaag gttcatatga atatttgact
     2581 catgaatcaa aggacgctat tgctaagaat aaacatatat acgacaaaaa agatattttg
     2641 aacattaatg attttgatat tgaccgctat ataacacttg atgaaagcca aaaaagaaaa
     2701 ttgaagaatt tacttttaga tatagtggat gactataatt tggtaaatac aaaagattta
     2761 atggctttta ttcgccttag gggagcggag tttggaattt taaatacgaa tgatgtaaaa
     2821 gatattgttt caacaaactc tagcgccttt agattatggt ttgagggcaa ttatcagtgt
     2881 ggatatagag caagttatgc aaaggttctt gatgctgaaa cgggggaaat aaaatgacaa
     2941 acaaagaaaa agagttattt gctgaaaatg aggaattaaa aaaagaaatt aaggacttaa
     3001 aagagcgtat tgaaagatac agagaaatgg aagttgaatt aagtacaaca atagatttat
     3061 tgagaggagg gattattgaa taaataaaag cccccctgac gaaagtccaa gggtttattg
     3121 ttttctaaaa tctgattacc aattagaatg aatatttccc aaatattaaa taataaaaca
     3181 aaaaaattaa aaaagtgttt ccaccatttt ttcaattttt ttataatttt tttaatctgt
     3241 tatttaaata gtttatagtt aaatttacat tttcattagt ccattcaata ttctctccaa
     3301 gataactacg aactgctaac aaaattctct ccctatgttc taatggagaa gattcagcca
     3361 ctgcatttcc cgcaatatct tttggtatga ttttacccgt gtccatagtt aaaatcatac
     3421 ggcataaagt taatatagag ttggtttcat catcctgata attatctatt aattcctctg
     3481 acgaatccat aatggctctt ctcacatcag aaaatggaat atcaggtagt aattcctcta
     3541 agtcataatt tccgtatatt cttttatttt ttcgttttgc ttggtaaagc attatggtta
     3601 aatctgaatt taattccttc tgaggaatgt atccttgttc ataaagctct tgtaaccatt
     3661 ctccataaat aaattcttgt ttgggaggat gattccacgg taccatttct tgctgaataa
     3721 taattgttaa ttcaatatat cgtaagttgc ttttatctcc tatttttttt gaaataggtc
     3781 taattttttg tataagtatt tctttacttt gatctgtcaa tggttcagat acgacgacta
     3841 aaaagtcaag atcactattt ggttttagtc cactctcaac tcctgatcca aacatgtaag
     3901 taccaataag gttatttttt aaatgtttcc gaagtatttt tttcacttta ttaatttgtt
     3961 cgtatgtatt caaatatatc ctcctcacta ttttgattag tacctatttt atatccatag
     4021 ttgttaatta aataaactta atttagttta tttatagatt tcattggctt ctaaattttt
     4081 tatctagata ataattattt tagttaattt tattctagat tatatatgat atgatctttc
     4141 atttccataa aactaaagta agtgtaaacc tattcattgt tttaaaaata tctcttgcca
     4201 gtcacgttac gttattagtt agttaccatc acggaaaaag gttatgctgc ttttaagacc
     4261 cactttcaca tttaagttgt ttttctaatc cgcatatgat caattcaagg ccgaataaga
     4321 aggctggctc tgcaccttgg tgatcaaata attcgatagc ttgtcgtaat aatggcggca
     4381 tactatcagt agtaggtgtt tccctttctt ctttagcgac ttgatgctct tgatcttcca
     4441 atacgcaacc taaagtaaaa tgccccacag cgctgagtgc atataatgca ttctctagtg
     4501 aaaaaccttg ttggcataaa aaggctaatt gattttcgag agtttcatac tgtttttctg
     4561 taggccgtgt acctaaatgt acttttgctc catcgcgatg acttagtaaa gcacatctaa
     4621 aacttttagc gttattacgt aaaaaatctt gccagctttc cccttctaaa gggcaaaagt
     4681 gagtatggtg cctatctaac atctcaatgg ctaaggcgtc gagcaaagcc cgcttatttt
     4741 ttacatgcca atacaatgta ggctgctcta cacctagctt ctgggcgagt ttacgggttg
     4801 ttaaaccttc gattccgacc tcattaagca gctctaatgc gctgttaatc actttacttt
     4861 tatctaatct agacatcatt aattcctcct ttttgttgac attatatcat tgatagagtt
     4921 atttgtcaaa ctagtttttt atttggatcc cctcgagttc atgaaaaact aaaaaaaata
     4981 ttgacactct atcattgata gagcataatt aaaataagct ctctatcatt gatagagtga
     5041 tatcgaattc ggacgcatag tggcctcaaa aatggataag aaatactcaa taggcttagc
     5101 tatcggcaca aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa
     5161 aaagttcaag gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc
     5221 tcttttattt gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag
     5281 aaggtataca cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat
     5341 ggcgaaagta gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga
     5401 caagaagcat gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga
     5461 gaaatatcca actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga
     5521 tttgcgctta atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat
     5581 tgagggagat ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca
     5641 aacctacaat caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc
     5701 gattctttct gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc
     5761 cggtgagaag aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc
     5821 taattttaaa tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac
     5881 ttacgatgat gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt
     5941 tttggcagct aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac
     6001 tgaaataact aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca
     6061 agacttgact cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat
     6121 cttttttgat caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga
     6181 agaattttat aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt
     6241 ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat
     6301 tccccatcaa attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta
     6361 tccattttta aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta
     6421 ttatgttggt ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga
     6481 agaaacaatt accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc
     6541 atttattgaa cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa
     6601 acatagtttg ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt
     6661 tactgaagga atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga
     6721 tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa
     6781 aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc
     6841 attaggtacc taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga
     6901 agaaaatgaa gatatcttag aggatattgt tttaacattg accttatttg aagataggga
     6961 gatgattgag gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca
     7021 gcttaaacgt cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat
     7081 tagggataag caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa
     7141 tcgcaatttt atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa
     7201 agcacaagtg tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag
     7261 ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt
     7321 aatggggcgg cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac
     7381 tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga
     7441 attaggaagt cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa
     7501 gctctatctc tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat
     7561 taatcgttta agtgattatg atgtcgatgc cattgttcca caaagtttcc ttaaagacga
     7621 ttcaatagac aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt
     7681 tccaagtgaa gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa
     7741 gttaatcact caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga
     7801 acttgataaa gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca
     7861 tgtggcacaa attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat
     7921 tcgagaggtt aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt
     7981 ccaattctat aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa
     8041 tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta
     8101 tggtgattat aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg
     8161 caaagcaacc gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat
     8221 tacacttgca aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg
     8281 agaaattgtc tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc
     8341 ccaagtcaat attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat
     8401 tttaccaaaa agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa
     8461 atatggtggt tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga
     8521 aaaagggaaa tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga
     8581 aagaagttcc tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt
     8641 taaaaaagac ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg
     8701 taaacggatg ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag
     8761 caaatatgtg aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga
     8821 agataacgaa caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat
     8881 tgagcaaatc agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt
     8941 tcttagtgca tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat
     9001 tcatttattt acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac
     9061 aattgatcgt aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca
     9121 atccatcact g
//
