LOCUS       sequence-422752-        8838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of an E. coli codon optimized fusion protein of 
            Cas9n-linker-M-MLV2 for prime editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8838)
  AUTHORS   Tong Y, Jorgensen TS, Whitford CM, Weber T, Lee SY
  TITLE     A versatile genetic engineering toolkit for E. coli based on 
            CRISPR-prime editing.
  JOURNAL   Nat Commun. 2021 Sep 1;12(1):5206. doi: 10.1038/s41467-021-25541-3.
  PUBMED    34471126
REFERENCE   2  (bases 1 to 8838)
  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 
            Commun."; date: "2021-09-1"; volume: "12(1):5206. doi"; pages: " 
            10.1038/s41467-021-25541-3"
COMMENT     Sequence Label: pCRISPR-PE-bacteria
FEATURES             Location/Qualifiers
     source          1..8838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        541..643
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             644..1303
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(710..729)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1324..1418
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1532..2077
                     /direction=RIGHT
                     /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     1978..1997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2194..2211
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2239..2266)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2282..2353)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(4513..6096,6097..6099,6100..8616))
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     RBS             8623..8634
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        8652..8707
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    8653..8671
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(8723..8838,1..511)
                     /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     complement(8799..8818)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga
       61 gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt
      121 tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa
      181 agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc
      241 ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg
      301 gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag
      361 ggaaacacct actactgata gtatgccgcc attattacga caagctatcg aattatttga
      421 tcaccaaggt gcagagccag ccttcttatt cggccttgaa ttgatcatat gcggattaga
      481 aaaacaactt aaatgtgaaa gtgggtctta agacgtcgat atctggcgaa aatgagacgt
      541 tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac taccgggcgt
      601 attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac
      661 tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca
      721 gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg catttttaaa
      781 gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct
      841 gatgaatgct catccggaat ttcgtatggc aatgaaagac ggtgagctgg tgatatggga
      901 tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg
      961 gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg
     1021 ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc
     1081 agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt
     1141 cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc
     1201 gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa
     1261 tgaattacaa cagtactgcg atgagtggca gggcggggcg taatttgata tcgagctcgc
     1321 ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa
     1381 cgctcggttg ccgccgggcg ttttttattg gtgagaatcc aagcactagt aacaacttat
     1441 atcgtatggg gctgacttca ggtgctacat ttgaagagat aaattgcact gaaatctaga
     1501 aatattttat ctgattaata agatgatctt cttgagatcg ttttggtctg cgcgtaatct
     1561 cttgctctga aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt ctctgagcta
     1621 ccaactcttt gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa cttgtccttt
     1681 cagtttagcc ttaaccggcg catgacttca agactaactc ctctaaatca attaccagtg
     1741 gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg actcaagacg atagttaccg
     1801 gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca tacagtccag cttggagcga
     1861 actgcctacc cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc ataacagcgg
     1921 aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc acgagggagc cgccaggggg
     1981 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg agcgtcagat
     2041 ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac ggctttgccg cggccctctc
     2101 acttccctgt taagtatctt cctggcatct tccaggaaat ctccgccccg ttcgtaagcc
     2161 atttccgctc gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga ggaagcggaa
     2221 tatatcccta ggtataaacg cagaaaggcc cacccgaagg tgagccagtg tgactctagt
     2281 agagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct
     2341 ttcgttttat ttgatgcctg gagatcctta ctcgagttat tacgggctgc tattttcaat
     2401 cagcagggtg ctggtatccg gggtttcggt aatcgccgct ttacgcgccg cttgatccgc
     2461 catgcgatta ccacgcgcct ccgcgctgtg acctttctgg tgacccgggc agtgaatgat
     2521 gctcagacgt ttcggcagga acagcgcttt cagcagcgcc agaatctcgt ccttattttt
     2581 gatttctttg ccttcgctgg tcagccaacc acgacgacgg tagatttcac catggatgtg
     2641 cgcggtcgca aacgcgtaac ggctatcggt ataaacattc agctttttgc cctccgccat
     2701 tttcagcgct tgggtcagcg caatcagctc cgcacgctgc gcgctggtac ccgccggcag
     2761 cgctttcgcc cagataactt cggtctcggt ggtaaccgcc gcacccgcct tacgctggcc
     2821 ttcttgcagc aggctgctgc cgtcggtata ccaggtgtga tccgcgtccg gcagcggctg
     2881 gtcggtcaga tccggacggg taccgtgcgc ctccgccaga atgtccaggc agttatgctg
     2941 cagaccttcc tccggcagcg gcagcagggt cgccgggttc agcgcaacca ccggaccgaa
     3001 ctgaacacgg tcggtatcca gcagcagcgc ttgatagtgg gtcatacgcg cgttgctcag
     3061 ccaacggtcc ggcggctgct taaccagcgc ttcaaccgca tgcggcgcca gaatcaccag
     3121 cggttgaccc atggtcagct tgcccgcgtc tttggtcaga accgcgatcg ccgcaaccat
     3181 acgcaggcac ggcggccagc ccgccgcaac cggatccagt ttcttgctca ggtacgcaac
     3241 cggacggcgc cacggaccca gtttctgggt cagaacgcct ttcgcataac cctgcttttc
     3301 atcaacaaac agctcgaacg gcttggtcag gtccggcaga cccagcgccg gcgcggtcag
     3361 cagcgcctgt ttgatctctt ggtacgcttt ctgctggtcc ggaccccagt taaacagggt
     3421 acccggtttg gtcagcgggt acagcggcgc cgccatttcc gcgaaacccg ggataaacag
     3481 acggcaaaaa cccgctttac ccaggaactc acgcagttgg cgcggggtct tcggggtcgg
     3541 ctgacccata acggtttcct tacgcgcctc ggtcagccag cgttgacctt ccttcagcag
     3601 gtagcccaga tacttcactt gcttctggca aatttgcgcc tttttcgcgc tcgcacggta
     3661 acccaggtta cccagggtct gcagcagcgc acgggtaccc tgttggcaat ccagttcgct
     3721 ggtcgccgcc agcagcagat cgtccacgta ctgcagcagg atcagatccg ggtgttggat
     3781 acggaagtcc gccagatcac ggtgcagcgc ctcgttgaac agggtcgggc tgtttttaaa
     3841 gccctgcggc agacgggtcc aggtcagttg gccgctgata cccatttccg ggtcacgcca
     3901 ttcaaacgcg aacagcggtt ggctggtcgg gtgcagacgc aggcagaaaa acgcgtcctt
     3961 cagatccagc acggtatacc actggtggct cggcggcaga ccgctcagca ggttgtacgg
     4021 gttcggaacg gtcggatgaa tatcttcaac gcgcttgtta acctcacgca ggtcttgcac
     4081 cggacggtaa tcattggtac ccggtttctt caccggcagc agcggggtat tccacgggct
     4141 ctggcacggc accagaatac cctggtccag cagacgttga atgtgcggct taatacccag
     4201 acgcgcttcc tggctcatcg gatactgctt gatgctaacc ggggtgctgg tcgccttcag
     4261 cggaatgatc agcggcgcct ggcgaaccgc cagacccatg ccgccggttt ccgcccacgc
     4321 ctgcggaaag tcgctcagcc aggtgctacc caggctaaca tccggctcct tgctggtctc
     4381 atgcagacga tactcgtcct cgatgttcag ggtgctgcta ccgccgctgc tgccgccgct
     4441 gctttccggg gtcgcgcttt cgctggtgcc cggggtttcg ctgccgctgc tgccgccgct
     4501 gctgccgccg ctgtcacctc ctagctgact caaatcaatg cgtgtttcat aaagaccagt
     4561 gatggattga tggataagag tggcatctaa aacttctttt gtagacgtat atcgtttacg
     4621 atcaattgtt gtatcaaaat atttaaaagc agcgggagct ccaagattcg tcaacgtaaa
     4681 taaatgaata atattttctg cttgttcacg tattggtttg tctctatgtt tgttatatgc
     4741 actaagaact ttatctaaat tggcatctgc taaaataaca cgcttagaaa attcactgat
     4801 ttgctcaata atctcatcta aataatgctt atgctgctcc acaaacaatt gtttttgttc
     4861 gttatcttct ggactaccct tcaacttttc ataatgacta gctaaatata aaaaattcac
     4921 atatttgctt ggcagagcca gctcatttcc tttttgtaat tctccggcac tagccagcat
     4981 ccgtttacga ccgttttcta actcaaaaag actatattta ggtagtttaa tgattaagtc
     5041 ttttttaact tccttatatc ctttagcttc taaaaagtca atcggatttt tttcaaagga
     5101 acttctttcc ataattgtga tccctagtaa ctctttaacg gattttaact tcttcgattt
     5161 ccctttttcc accttagcaa ccactaggac tgaataagct accgttggac tatcaaaacc
     5221 accatatttt tttggatccc agtctttttt acgagcaata agcttgtccg aatttctttt
     5281 tggtaaaatt gactccttgg agaatccgcc tgtctgtact tctgttttct tgacaatatt
     5341 gacttggggc atggacaata ctttgcgcac tgtggcaaaa tctcgccctt tatcccagac
     5401 aatttctcca gtttccccat tagtttcgat tagagggcgt ttgcgaatct ctccatttgc
     5461 aagtgtaatt tctgttttga agaagttcat gatattagag taaaagaaat attttgcggt
     5521 tgctttgcct atttcttgct cagacttagc aatcatttta cgaacatcat aaactttata
     5581 atcaccatag acaaactccg attcaagttt tggatatttc ttaatcaaag cagttccaac
     5641 gacggcattt agatacgcat catgggcatg atggtaattg ttaatctcac gtactttata
     5701 gaattggaaa tcttttcgga agtcagaaac taatttagat tttaaggtaa tcactttaac
     5761 ctctcgaata agtttatcat tttcatcgta tttagtattc atgcgactat ccaaaatttg
     5821 tgccacatgc ttagtgattt ggcgagtttc aaccaattgg cgtttgataa aaccagcttt
     5881 atcaagttca ctcaaacctc cacgttcagc tttcgttaaa ttatcaaact tacgttgagt
     5941 gattaacttg gcgtttagaa gttgtctcca atagtttttc atctttttga ctacttcttc
     6001 acttggaacg ttatccgatt taccacgatt tttatcagaa cgcgttaaga ccttattgtc
     6061 tattgaatcg tctttaagga aactttgtgg aacaatggca tcgacatcat aatcacttaa
     6121 acgattaata tctaattctt ggtccacata catgtctctt ccattttgga gataatagag
     6181 atagagcttt tcattttgca attgagtatt ttcaacagga tgctctttaa gaatctgact
     6241 tcctaattct ttgatacctt cttcgattcg tttcatacgc tctcgcgaat ttttctggcc
     6301 cttttgagtt gtctgatttt cacgtgccat ttcaataacg atattttctg gcttatgccg
     6361 ccccattact ttgaccaatt catcaacaac ttttacagtc tgtaaaatac cttttttaat
     6421 agcagggcta ccagctaaat ttgcaatatg ttcatgtaaa ctatcgcctt gtccagacac
     6481 ttgtgctttt tgaatgtctt ctttaaatgt caaactatca tcatggatca gctgcataaa
     6541 attgcgattg gcaaaaccat ctgatttcaa aaaatctaat attgttttgc cagattgctt
     6601 atccctaata ccattaatca attttcgaga caaacgtccc caaccagtat aacggcgacg
     6661 tttaagctgt ttcatcacct tatcatcaaa gaggtgagca tatgttttaa gtctttcctc
     6721 aatcatctcc ctatcttcaa ataaggtcaa tgttaaaaca atatcctcta agatatcttc
     6781 attttcttca ttatccaaaa aatctttatc tttaataatt tttagcaaat catggtaggt
     6841 acctaatgaa gcattaaatc tatcttcaac tcctgaaatt tcaacactat caaaacattc
     6901 tatttttttg aaataatctt cttttaattg cttaacggtt acttttcgat ttgttttgaa
     6961 gagtaaatca acaatggctt tcttctgttc acctgaaaga aatgctggtt ttcgcattcc
     7021 ttcagtaaca tatttgacct ttgtcaattc gttataaacc gtaaaatact cataaagcaa
     7081 actatgtttt ggtagtactt tttcatttgg aagattttta tcaaagtttg tcatgcgttc
     7141 aataaatgat tgagctgaag cacctttatc gacaacttct tcaaaattcc atggggtaat
     7201 tgtttcttca gacttccgag tcatccatgc aaaacgacta ttgccacgcg ccaatggacc
     7261 aacataataa ggaattcgaa aagtcaagat tttttcaatc ttctcacgat tgtcttttaa
     7321 aaatggataa aagtcttctt gtcttctcaa aatagcatgc agctcaccca agtgaatttg
     7381 atggggaata gagccgttgt caaaggtccg ttgcttgcgc agcaaatctt cacgatttag
     7441 tttcaccaat aattcctcag taccatccat tttttctaaa attggtttga taaatttata
     7501 aaattcttct tggctagctc ccccatcaat ataacctgca tatccgtttt ttgattgatc
     7561 aaaaaagatt tctttatact tttctggaag ttgttgtcga actaaagctt ttaaaagagt
     7621 caagtcttga tgatgttcat cgtagcgttt aatcattgaa gctgataggg gagccttagt
     7681 tatttcagta tttactctta ggatatctga aagtaaaata gcatctgata aattcttagc
     7741 tgccaaaaac aaatcagcat attgatctcc aatttgcgcc aataaattat ctaaatcatc
     7801 atcgtaagta tcttttgaaa gctgtaattt agcatcttct gccaaatcaa aatttgattt
     7861 aaaattaggg gtcaaaccca atgacaaagc aatgagattc ccaaataagc catttttctt
     7921 ctcaccgggg agctgagcaa tgagattttc taatcgtctt gatttactca atcgtgcaga
     7981 aagaatcgct ttagcatcta ctccacttgc gttaataggg ttttcttcaa ataattgatt
     8041 gtaggtttgt accaactgga taaatagttt gtccacatca ctattatcag gatttaaatc
     8101 tccctcaatc aaaaaatgac cacgaaactt aatcatatgc gctaaggcca aatagattaa
     8161 gcgcaaatcc gctttatcag tagaatctac caattttttt cgcagatgat agatagttgg
     8221 atatttctca tgataagcaa cttcatctac tatatttcca aaaataggat gacgttcatg
     8281 cttcttgtct tcttccacca aaaaagactc ttcaagtcga tgaaagaaac tatcatctac
     8341 tttcgccatc tcatttgaaa aaatctcctg tagataacaa atacgattct tccgacgtgt
     8401 ataccttcta cgagctgtcc gtttgagacg agtcgcttcc gctgtctctc cactgtcaaa
     8461 taaaagagcc cctataagat tttttttgat actgtggcgg tctgtatttc ccagaacctt
     8521 gaacttttta gacggaacct tatattcatc agtgatcacc gcccatccga cgctatttgt
     8581 gccgatatct aagcctattg agtatttctt atccatagat cctttctcct ctttagatct
     8641 tttgaattct tttctctatc actgataggg agtggtaaaa taactctatc aacgatagag
     8701 tgtcaacaaa aattaggaat taatgatgtc tagattagat aaaagtaaag tgattaacag
     8761 cgcattagag ctgcttaatg aggtcggaat cgaaggttta acaacccgta aactcgccca
     8821 gaagctaggt gtagagca
//
