LOCUS       sequence-351818-        5465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis plasmid with constitutive bacterial expression of DddI 
            immunity protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5465)
  AUTHORS   Mok BY, Kotrys AV, Raguram A, Huang TP, Mootha VK, Liu DR
  TITLE     CRISPR-free base editors with enhanced activity and expanded 
            targeting scope in mitochondrial and nuclear DNA.
  JOURNAL   Nat Biotechnol. 2022 Sep;40(9):1378-1387. doi: 
            10.1038/s41587-022-01256-8. Epub 2022 Apr 4.
  PUBMED    35379961
REFERENCE   2  (bases 1 to 5465)
  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"; doi: 
            "10.1038/s41587-022-01256-8"; journalName: "Nat Biotechnol"; date: 
            "2022-09"; volume: "40"; issue: "9"; pages: "1378-1387"
COMMENT     Sequence Label: pJC175e-DddI
FEATURES             Location/Qualifiers
     source          1..5465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(36..986)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      1034..1256
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             2014..3288
                     /codon_start=1
                     /gene="III"
                     /product="pIII"
                     /label=M13 gene III
                     /note="minor coat protein"
                     /translation="MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKTL
                     DRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGG
                     TKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQ
                     GALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQ
                     GQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKM
                     ANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGS
                     NSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVF
                     AFLLYVATFMYVFSTFANILRNKES"
     CDS             complement(3399..3431)
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     terminator      complement(4008..4035)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4127..4213)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4366..4383
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(join(4429..5220,5221..5289))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5052..5071
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5290..5394)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(5462..5465,1..15)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggcgtatcac gaggccctta ggccaaatag gccgttcaga tccttccgta tttagccagt
       61 atgttctcta gtgtggttcg ttgtttttgc gtgagccatg agaacgaacc attgagatca
      121 tgcttacttt gcatgtcact caaaaatttt gcctcaaaac tggtgagctg aatttttgca
      181 gttaaagcat cgtgtagtgt ttttcttagt ccgttacgta ggtaggaatc tgatgtaatg
      241 gttgttggta ttttgtcacc attcattttt atctggttgt tctcaagttc ggttacgaga
      301 tccatttgtc tatctagttc aacttggaaa atcaacgtat cagtcgggcg gcctcgctta
      361 tcaaccacca atttcatatt gctgtaagtg tttaaatctt tacttattgg tttcaaaacc
      421 cattggttaa gccttttaaa ctcatggtag ttattttcaa gcattaacat gaacttaaat
      481 tcatcaaggc taatctctat atttgccttg tgagttttct tttgtgttag ttcttttaat
      541 aaccactcat aaatcctcat agagtatttg ttttcaaaag acttaacatg ttccagatta
      601 tattttatga atttttttaa ctggaaaaga taaggcaata tctcttcact aaaaactaat
      661 tctaattttt cgcttgagaa cttggcatag tttgtccact ggaaaatctc aaagccttta
      721 accaaaggat tcctgatttc cacagttctc gtcatcagct ctctggttgc tttagctaat
      781 acaccataag cattttccct actgatgttc atcatctgag cgtattggtt ataagtgaac
      841 gataccgtcc gttctttcct tgtagggttt tcaatcgtgg ggttgagtag tgccacacag
      901 cataaaatta gcttggtttc atgctccgtt aagtcatagc gactaatcgc tagttcattt
      961 gctttgaaaa caactaattc agacatacat ctcaattggt ctaggtgatt ttaatcacta
     1021 taccaattga gatgggctag tcaatgataa ttactagtcc ttttcctttg agttgtgggt
     1081 atctgtaaat tctgctagac ctttgctgga aaacttgtaa attctgctag accctctgta
     1141 aattccgcta gacctttgtg tgtttttttt gtttatattc aagtggttat aatttataga
     1201 ataaagaaag aataaaaaaa gataaaaaga atagatccca gccctgtgta taactcacta
     1261 ctttagtcag ttccgcagta ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa
     1321 aacagacctt aaaaccctaa aggcttaagt agcaccctcg caagctcggg caaatcgctg
     1381 aatattcctt ttgtctccga ccatcaggca cctgagtcgc tgtctttttc gtgacattca
     1441 gttcgctgcg ctcacggctc tggcagtgaa tgggggtaaa tggcactaca ggcgcctttt
     1501 atggattcat gcaaggaaac tacccataat acaagaaaag cccgtcacgg gcttctcagg
     1561 gcgttttatg gcgggtctgc tatgtggtgc tatctgactt tttgctgttc agcagttcct
     1621 gccctctgat tttccagtct gaccacttcg gattatcccg tgacaggtca ttcagactgg
     1681 ctaatgcacc cagtaaggca gcggtatcat caacaggctt acccgtctta ctgtcaagag
     1741 gacatccggt caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttgctga
     1801 ggagacttag ggaccctgat gaaattcgcc acttgttagt gtaattcgct aactcatcct
     1861 ggcatgttgc tgttgattct tcaatcagat ctttataaat caaaaagata aaaaattggc
     1921 acgcaaattg tattaacagt tcagcaggac aatcctgaac gcagaaatca agaggacaac
     1981 attacctgca ggtgcagtaa ggaggaaaaa aaaatgaaaa aattattatt cgcaattcct
     2041 ttagttgttc ctttctattc tcactccgct gaaactgttg aaagttgttt agcaaaaccc
     2101 catacagaaa attcatttac taacgtctgg aaagacgaca aaactttaga tcgttacgct
     2161 aactatgagg gctgtctgtg gaatgctaca ggcgttgtag tttgtactgg tgacgaaact
     2221 cagtgttacg gtacatgggt tcctattggg cttgctatcc ctgaaaatga gggtggtggc
     2281 tctgagggtg gcggttctga gggtggcggt tctgagggtg gcggtactaa acctcctgag
     2341 tacggtgata cacctattcc gggctatact tatatcaacc ctctcgacgg cacttatccg
     2401 cctggtactg agcaaaaccc cgctaatcct aatccttctc ttgaggagtc tcagcctctt
     2461 aatactttca tgtttcagaa taataggttc cgaaataggc agggggcatt aactgtttat
     2521 acgggcactg ttactcaagg cactgacccc gttaaaactt attaccagta cactcctgta
     2581 tcatcaaaag ccatgtatga cgcttactgg aacggtaaat tcagagactg cgctttccat
     2641 tctggcttta atgaggatcc attcgtttgt gaatatcaag gccaatcgtc tgacctgcct
     2701 caacctcctg tcaatgctgg cggcggctct ggtggtggtt ctggtggcgg ctctgagggt
     2761 ggtggctctg agggtggcgg ttctgagggt ggcggctctg agggaggcgg ttccggtggt
     2821 ggctctggtt ccggtgattt tgattatgaa aagatggcaa acgctaataa gggggctatg
     2881 accgaaaatg ccgatgaaaa cgcgctacag tctgacgcta aaggcaaact tgattctgtc
     2941 gctactgatt acggtgctgc tatcgatggt ttcattggtg acgtttccgg ccttgctaat
     3001 ggtaatggtg ctactggtga ttttgctggc tctaattccc aaatggctca agtcggtgac
     3061 ggtgataatt cacctttaat gaataatttc cgtcaatatt taccttccct ccctcaatcg
     3121 gttgaatgtc gcccttttgt ctttggcgct ggtaaacctt acgagttcag tatcgactgc
     3181 gataagatca acctgttccg cggtgtcttt gcgtttcttt tatatgttgc cacctttatg
     3241 tatgtatttt ctacgtttgc taacatactg cgtaataagg agtcttaacg gagccaatgt
     3301 acgcaaaaaa tcccgcttca gcggggtttt ttcgccaaaa aaaaccccgc cctgtcaggg
     3361 gcggggtttt tttttagggc cctactgact gttcattatt ttcctaatct attcatttca
     3421 atatctgtat atctagacag ttcgctccag gtcagctgct ccggacgctg tttggtcgca
     3481 aagaactgct tcgcggcttc aatcatctgc tctttgctca ccacgcaatc cttgctcagg
     3541 tccacttcac cgcccagttt gttttcataa aacttctcgg taccgccgct ggcgctgtta
     3601 cccttgctca cgaagttctc gccgatatcc atatagtaca ccacagcaat gtcgttcttc
     3661 gcaaagatgt tcagctgcgg gaaaccgctt tcctcaaagg tcagcacgaa gttgttggca
     3721 tcaaaagccg ggcgacggct caggaattcc accaggctat ccacctcgtc aatttcgatc
     3781 tcgccgtcga aatcgtcagc gtacattttt ttttcctcct tactctagta aaagttaaac
     3841 aaaattattt gtagagggaa accgttgtgg tctccctgaa tatattatac gagccttatg
     3901 catgcccgta aagttatcca gcaaccactc atagacctag ggcagcagat agggacgacg
     3961 tggtgttagc tgtgctctag aatcctcagc gagtttgtag aaacgcaaaa aggccatccg
     4021 tcaggatggc cttctgctta atttgatgcc tggcagttta tggcgggcgt cctgcccgcc
     4081 accctccggg ccgttgcttc gcaacgttca aatccgctcc cggcggattt gtcctactca
     4141 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct
     4201 ttcgttttat ttgatgcctg gcagttccct actctcgcat ggggagaccc cacactacca
     4261 tcggcgctac ggcgtttcac ttctgagttc ggcatggggt caggtgggac caccgcgcta
     4321 ctgccgccag gcaaattctg ttttatcaga ccgcttctgc gttctgattt aatctgtatc
     4381 aggctgaaaa tcttctctca tccgccaaaa cagccatgcc catggcgttt accaatgctt
     4441 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4501 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4561 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4621 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4681 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4741 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4801 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4861 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4921 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4981 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5041 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5101 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5161 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5221 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5281 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5341 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5401 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     5461 aaata
//
