LOCUS       sequence-497000-        8469 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  To express the EPEC DarT2(G49D)-nScCas9(D10A) fusion protein in 
            experiments for Append Editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8469)
  AUTHORS   Gupta D, Patinios C, Bassett HV, Kibe A, Collins SP, Kamm C, Wang Y,
            Zhao C, Vollen K, Toussaint C, Calvin I, Cullot G, Aird EJ, Polkoff 
            KM, Nguyen-Vo TP, Migur A, Schut F, Al'Abri IS, Achmedov T, Del Re 
            A, Corn JE, Saliba AE, Crook N, Stepanova AN, Alonso JM, Beisel CL
  TITLE     Targeted DNA ADP-ribosylation triggers templated repair in bacteria 
            and base mutagenesis in eukaryotes.
  JOURNAL   Nat Biotechnol. 2025 Sep 4. doi: 10.1038/s41587-025-02802-w.
  PUBMED    40908325
REFERENCE   2  (bases 1 to 8469)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 13, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 Sep 4. doi: 10.1038/s41587-025-02802-w."
COMMENT     Sequence Label: pCBS-6738
FEATURES             Location/Qualifiers
     source          1..8469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     386..405
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(585..1463)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     primer_bind     1697..1716
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     terminator      6754..6825
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6841..6868
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(6946..7605)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KNVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7520..7539
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7606..7708)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(8100..8117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(8234..8469,1..310))
                     /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(8314..8333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcacgaac cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag
       61 tccaacccgg aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga
      121 ggagttagtc ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga
      181 ctgcgctcct ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga
      241 aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa
      301 acgatctcaa gaagatcatc ttattaatca gataaaatat ttctaggctc atgagcccga
      361 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      421 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatctgc tcatgtttga
      481 cagcttatca tcgatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc
      541 ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg
      601 gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg
      661 cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg
      721 gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc
      781 tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac
      841 ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga
      901 tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg
      961 ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc
     1021 gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc
     1081 ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta
     1141 agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga
     1201 gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc
     1261 ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga
     1321 ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg
     1381 gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg
     1441 ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat
     1501 tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc
     1561 aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca
     1621 aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac
     1681 ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga
     1741 cgctttttat cgcaactctc tactgtttct cctgcatgcc acttaaaaag gagatcaaca
     1801 atgaaagcaa ttttcgtact gaaacatctt aatcatgcta aggagaaata ctagtatggc
     1861 ctatgattac tcagccagct tgaatccaca gaaagctctt atctggcgca ttgtccatcg
     1921 tgataacatt ccttggatct tagacaatgg tctgcactgc ggtaatagct tggttcaagc
     1981 cgagaactgg attaacatcg acaatcccga gttgattggt aaacgcgctg ggcacccggt
     2041 tcccgtaggc accgggggta cgttgcacga ttatgtcccg ttctacttta cgccttttag
     2101 tcctatgctt atgaatatcc attcaggccg cggcggcatt aaacgccgcc ccaatgaaga
     2161 gattgtaatc ttagttagca acttacgcaa cgtcgcagcc catgacgtgc cctttgtttt
     2221 taccgactca cacgcctatt ataattggac aaactactat acgtcgttaa actcattgga
     2281 ccagatcgat tggccaattt tgcaagcccg cgatttccgc cgtgacccag atgacccggc
     2341 taagtttgaa cgctaccaag cagaggcctt aatctggcaa cactgtccga tctcattact
     2401 tgacggtatc atctgttact cggaagaggt ccgcttgcag ttggagcagt ggttgtttca
     2461 acgtaatttg acaatgtcag tacacacccg ttctggctgg tatttttcga gtggcggatc
     2521 gtctggtggc agttcaggct cggaaacacc aggcacaagc gagtccgcga cacccgaatc
     2581 gagtggaggg agctccgggg gttccgagaa gaaatattct atcggtctgg ccatcggtac
     2641 gaactcggta ggttgggcag tgattaccga tgactataaa gtcccatcaa agaaattcaa
     2701 ggtgctgggg aacacgaacc gcaagagcat taaaaaaaac ttaatgggcg cccttctttt
     2761 tgattcgggg gaaacagcgg aggcgacacg cttaaaacgc actgcccgcc gccgttacac
     2821 gcgtcgcaaa aatcgcatcc gttaccttca agaaatcttt gccaatgaga tggcaaaact
     2881 ggatgactct tttttccaac gccttgaaga gtctttcctt gtcgaagagg ataagaaaaa
     2941 cgagcgtcat cctattttcg gaaatcttgc agacgaggtg gcttaccacc gtaactatcc
     3001 tactatctac catttgcgca aaaagctggc ggactcacca gaaaaggcag acttacgtct
     3061 gatttatctg gcacttgctc acattatcaa gttccgcggg cactttttga tcgagggaaa
     3121 gttgaatgca gagaattcag atgtagcgaa acttttttat caactgattc aaacctataa
     3181 tcagctgttt gaagaaagtc cacttgacga aatcgaggta gatgcaaaag ggattttgtc
     3241 agcacgtctt tcaaagtcta agcgtttgga aaagcttatc gctgttttcc ccaacgagaa
     3301 aaagaacggg cttttcggca acattattgc attagcattg ggcctgactc cgaatttcaa
     3361 atctaatttt gacttaacag aagatgcaaa attgcaatta agcaaggata catatgacga
     3421 cgatcttgac gagcttctgg gccaaatcgg agatcagtac gccgaccttt ttagcgcagc
     3481 taagaactta tcggatgcca ttttattatc tgacattttg cgtagcaatt cggaagtaac
     3541 gaaggctcct ctgagtgcct cgatggttaa gcgctacgat gaacaccatc aggatttggc
     3601 ccttttgaaa acactggttc gtcaacagtt cccagagaaa tatgcggaga tctttaagga
     3661 cgataccaaa aatggttacg caggatacgt aggtattgga attaaacacc gtaagcgtac
     3721 cacaaagttg gccacccaag aggaattcta taaatttatc aagccgatct tggaaaaaat
     3781 ggacggggct gaagaattgc tggcaaagct gaaccgcgat gatcttttgc gcaagcaacg
     3841 taccttcgac aatggatcga ttccacatca aattcatctg aaggaacttc acgcgatttt
     3901 acgtcgtcag gaggagttct accctttctt gaaagaaaat cgtgaaaaaa ttgaaaagat
     3961 tttaacgttc cgcatcccat attacgtagg gcccttagca cgcggtaact cacgtttcgc
     4021 ttggcttaca cgtaaatcgg aggaagcaat tacaccatgg aattttgaag aagtagtaga
     4081 taagggcgcc tccgcacaga gttttattga gcgtatgacc aatttcgatg agcaactgcc
     4141 aaataagaag gtactgccta agcactcgtt actttacgaa tactttacgg tctacaacga
     4201 attgacaaaa gtaaaatacg ttactgaacg tatgcgtaag ccggaattct tatcagggga
     4261 acagaagaag gctattgttg atttattgtt taagacaaat cgtaaagtaa ctgtgaagca
     4321 gcttaaagaa gactacttta agaagattga atgttttgac tctgtggaga tcattggggt
     4381 agaggaccgt tttaatgctt ctctgggcac gtatcatgac ttattaaaga tcattaaaga
     4441 caaggatttt ttagataatg aggaaaatga agatattctg gaggacatcg tcttaacact
     4501 tactttgttc gaagatcgtg aaatgattga agaacgtctg aaaacctatg ctcatttgtt
     4561 tgacgacaaa gtcatgaaac agctgaaacg ccgccattat acgggctggg gccgtctgtc
     4621 tcgcaaaatg attaacggca ttcgcgacaa gcagagcgga aagacaatcc tggatttcct
     4681 gaagagcgat ggcttctcta accgtaattt catgcaatta atccacgacg attccttgac
     4741 ctttaaggag gagattgaaa aggctcaggt gagcgggcag ggggattcgc ttcatgagca
     4801 gatcgcggat ttggccggct ctccagcaat taagaaagga atcctgcaaa ctgtcaagat
     4861 cgtagatgaa cttgtaaaag taatggggca caagccagaa aacattgtaa tcgagatggc
     4921 ccgcgaaaac cagacgacaa cgaaggggtt acaacagagt cgtgaacgca agaaacgtat
     4981 tgaagaaggg attaaggaat tggaatcgca gattttgaaa gagaaccccg tggaaaatac
     5041 acagctgcaa aatgagaaac tgtaccttta ttatttacag aacggccgcg atatgtatgt
     5101 agatcaagaa ttggatatta accgtttgtc agattatgac gtcgatcata tcgttccgca
     5161 gtcgtttatc aaagacgact caattgacaa caaggtgctg actcgttcgg tcgagaaccg
     5221 tgggaaaagc gataatgtac cttcagaaga ggttgtaaaa aagatgaaaa actattggcg
     5281 ccaattatta aacgctaaac tgattacaca acgcaagttc gataatttaa caaaagccga
     5341 gcgcgggggg ttgtctgagg cggataaggc aggcttcatt aagcgccaat tggtcgagac
     5401 acgtcaaatc accaaacatg tagctcgcat tttagactcc cgtatgaata cgaagcgtga
     5461 caagaacgat aaacccatcc gtgaagtaaa ggtcattact ttaaaaagta aacttgtttc
     5521 ggactttcgt aaagattttc aactgtataa ggttcgtgat atcaataatt atcatcatgc
     5581 ccatgacgct tacttgaacg cggtcgtcgg taccgcatta attaaaaagt accccaaatt
     5641 ggaatctgag tttgtctacg gcgattataa ggtgtatgac gtacgtaaga tgatcgctaa
     5701 aagcgaacaa gagattggga aggcaactgc gaaacgtttt ttctatagta atattatgaa
     5761 ttttttcaaa accgaagtga agttagccaa cggcgagatt cgtaaacgcc cgctgatcga
     5821 aaccaacgga gaaacgggag aagtagtatg gaataaagag aaggattttg ctactgttcg
     5881 caaggtgtta gccatgcctc aggttaacat cgttaagaag accgaggtcc agacaggagg
     5941 gttttccaaa gaaagcattt tgtcaaagcg cgaaagtgct aaactgatcc cgcgcaaaaa
     6001 gggctgggac acccgtaaat acggagggtt tgggtccccc actgttgctt acagcattct
     6061 tgtagtagcc aaggttgaga agggcaaagc taagaaattg aagagtgtga aagttttagt
     6121 aggaattact atcatggaaa agggcagcta tgaaaaggac cctattggtt ttctggaggc
     6181 taaaggatac aaggacatta aaaaagaact gatctttaag ttaccgaagt attccttatt
     6241 tgaacttgag aatgggcgtc gccgcatgct ggctagcgct acggagctgc agaaggctaa
     6301 cgaattagtg cttcctcaac atttagtgcg cctgttatac tacactcaga atatcagcgc
     6361 tacaacaggg tctaacaatt taggttacat tgaacaacat cgcgaggagt tcaaggaaat
     6421 tttcgaaaaa atcattgatt tttcggagaa atacatcttg aagaacaaag ttaactctaa
     6481 tttaaagtct agctttgatg aacaattcgc tgtaagtgat agcattcttc tttccaattc
     6541 ttttgtgtca ttattgaaat ataccagctt cggtgcgtcg ggaggattca cgtttcttga
     6601 ccttgacgtc aaacaggggc gtttacgtta tcaaactgtc acagaagtgt tggatgccac
     6661 tctgatttat cagtcaatca cgggtttata cgagactcgc acagacctta gtcagctggg
     6721 aggagactaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc tcagtcgaaa
     6781 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact
     6841 ggctcacctt cgggtgggcc tttctgcgtt tatacctagg gagacatcac ttattcaggc
     6901 gtagcaacca ggcgtttaag ggcaccaata actgccttaa aaaaattacg ccccgccctg
     6961 ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca
     7021 aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata
     7081 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa
     7141 actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt
     7201 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa
     7261 ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg
     7321 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc
     7381 catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata
     7441 aaacttgtgc ttatttttct ttacgttctt taaaaaggcc gtaatatcca gctgaacggt
     7501 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca
     7561 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc
     7621 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
     7681 aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg
     7741 cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac
     7801 aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttactga tttagtgtat
     7861 gatggtgttt ttgaggtgct ccagtggctt ctgtttctat cagctgtccc tcctgttcag
     7921 ctactgacgg ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg
     7981 tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc atgtggcagg
     8041 agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat acaggatata ttccgcttcc
     8101 tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa
     8161 cggggcggag atttcctgga agatgccagg aagatactta acagggaagt gagagggccg
     8221 cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct
     8281 caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg
     8341 gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat
     8401 ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag
     8461 ctggactgt
//
