LOCUS       22l-COX8-3xFlag-TALE(RNR2-Right)-TadA(8e+V106W)       6040 bp    DNA     circular SYN 14-APR-2025
DEFINITION  mitoBE expression vector, target mtRNR2 gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6040)
  AUTHORS   Yi Z, Zhang X, Tang W, Yu Y, Wei X, Zhang X, Wei W
  TITLE     Strand-selective base editing of human mitochondrial DNA using 
            mitoBEs.
  JOURNAL   Nat Biotechnol. 2023 May 22. doi: 10.1038/s41587-023-01791-y.
  PUBMED    37217751
REFERENCE   2  (bases 1 to 6040)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2023 May 22. doi: 10.1038/s41587-023-01791-y."
COMMENT     Sequence Label: 22l-COX8-3xFlag-TALE(RNR2-Right)-TadA(8e+V106W)
FEATURES             Location/Qualifiers
     source          1..6040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      421..430
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             427..504
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MASVLTPLLLRGLTGSARRLPVPRAK"
     CDS             517..582
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3396..3412)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3396..3412)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3409..3431)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3420..3436
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3444..3450,3451..3468,3469..3474))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3489..3510
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3627..3644)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3798..4386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3878..3897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4557..5348,5349..5417))
                     /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     5180..5199
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5418..5522)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5601..5620)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5792..6040,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagac ccaagctggc tagcaccgcc
      421 gccaccatgg cctccgttct gaccccgctg ctgctgcgtg gcctgaccgg ctccgctcgt
      481 cgtctgccag ttccgcgtgc gaaaatccat tccctggact acaaagacca tgacggtgat
      541 tataaagatc atgacatcga ttacaaggat gacgatgaca agatggacat cgcggacctc
      601 aggacgctgg gctacagcca gcaacagcaa gagaagatca agcccaaagt aaggagcacc
      661 gtggcccagc accacgaggc cctggtgggt cacggcttca cccacgcgca tatcgttgct
      721 ctgagccaac atcccgcagc tctgggtacc gttgcggtga agtatcagga catgatcgcg
      781 gcactgcctg aagctacaca cgaagccata gtgggcgttg gcaagcagtg gagcggtgcc
      841 agagcgcttg aggcactgtt gacggtggct ggcgagctga ggggaccgcc actgcaactg
      901 gacaccggcc aactgctgaa gatcgccaag aggggaggcg tgacggcggt ggaggccgtg
      961 catgcctgga ggaacgcact gaccggcgcg cccctgaacc tgacaccaga gcaagtagtg
     1021 gctattgcaa gtaacaacgg tggcaaacaa gcgctggaga ccgtgcagag gctccttccg
     1081 gtgctctgcc aagcacacgg tctcactccg gaacaggtgg tcgcaatcgc gagccacgac
     1141 ggcggcaagc aagcccttga gacagtccaa agacttttgc ctgtcctttg tcaggcgcat
     1201 ggccttacgc ctgagcaagt cgttgcgatc gcctccaaca tcggcggaaa acaggctttg
     1261 gaaaccgtgc agcggttgct gcccgttttg tgccaagccc acggattgac ccccgaacag
     1321 gttgtagcca tagcttctca cgacggaggt aagcaggcac tggaaaccgt gcagcgcctg
     1381 ctcccagtac tgtgtcaggc tcatgggctg acaccagagc aagtagtggc tattgcaagt
     1441 cacgacggtg gcaaacaagc gctggagacc gtgcagaggc tccttccggt gctctgccaa
     1501 gcacacggtc tcactccgga acaggtggtc gcaatcgcga gcaacatcgg cggcaagcaa
     1561 gcccttgaga cagtccaaag acttttgcct gtcctttgtc aggcgcatgg ccttacgcct
     1621 gagcaagtcg ttgcgatcgc ctccaacggc ggcggaaaac aggctttgga aaccgtgcag
     1681 cggttgctgc ccgttttgtg ccaagcccac ggattgaccc ccgaacaggt tgtagccata
     1741 gcttctcacg acggaggtaa gcaggcactg gaaaccgtgc agcgcctgct cccagtactg
     1801 tgtcaggctc atgggctcac tccggaacag gtggtcgcaa tcgcgagcaa caacggcggc
     1861 aagcaagccc ttgagacagt ccaaagactt ttgcctgtcc tttgtcaggc gcatggcctt
     1921 acgcctgagc aagtcgttgc gatcgcctcc aacaacggcg gaaaacaggc tttggaaacc
     1981 gtgcagcggt tgctgcccgt tttgtgccaa gcccacggat tgacccccga acaggttgta
     2041 gccatagctt ctaacaacgg aggtaagcag gcactggaaa ccgtgcagcg cctgctccca
     2101 gtactgtgtc aggctcatgg gctgacacca gagcaagtag tggctattgc aagtaacatc
     2161 ggtggcaaac aagcgctgga gaccgtgcag aggctccttc cggtgctctg ccaagcacac
     2221 ggtctcactc cggaacaggt ggtcgcaatc gcgagcaacg gcggcggcaa gcaagccctt
     2281 gagacagtcc aaagactttt gcctgtcctt tgtcaggcgc atggccttac gcctgagcaa
     2341 gtcgttgcga tcgcctccaa caacggcgga aaacaggctt tggaaaccgt gcagcggttg
     2401 ctgcccgttt tgtgccaagc ccacggattg acccccgaac aggttgtagc catagcttct
     2461 aacggcggag gtaagcaggc actggaaagc atcgtggccc agctgagcag acccgaccct
     2521 gccctcgccg ctctgaccaa cgaccacctg gtggccctgg cttgcctcgg gggcaggcca
     2581 gctcttgacg ccgtgaagaa gggccttggt ggatcctctg aggtggagtt ttcccacgag
     2641 tactggatga gacatgccct gaccctggcc aagagggcac gcgatgagag ggaggtgcct
     2701 gtgggagccg tgctggtgct gaacaataga gtgatcggcg agggctggaa cagagccatc
     2761 ggcctgcacg acccaacagc ccatgccgaa attatggccc tgagacaggg cggcctggtc
     2821 atgcagaact acagactgat tgacgccacc ctgtacgtga cattcgagcc ttgcgtgatg
     2881 tgcgccggcg ccatgatcca ctctaggatc ggccgcgtgg tgtttggctg gaggaactcc
     2941 aaaaggggcg ccgcaggctc cctgatgaac gtgctgaatt accccggcat gaatcaccgc
     3001 gtcgaaatta ccgagggaat cctggcagat gaatgtgccg ccctgctgtg cgatttctac
     3061 cggatgccta gacaggtgtt caatgctcag aagaaggccc agagctccat caattgataa
     3121 ggggtctttg tcctctgtac tgtctctctc cttgccccta acccaaaaag cttcattttt
     3181 ctgtgtaggc tgcacaagag ccttgattga agatatattc tttctgaaca gtatttaagg
     3241 tttccaataa aatgtacacc cctcagaaac cggtgtttaa acgaagacaa tagcaggcat
     3301 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata
     3361 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3421 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag
     3481 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3541 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3601 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3661 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3721 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3781 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3841 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3901 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3961 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4021 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4081 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4141 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4201 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4261 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4321 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4381 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4441 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4501 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4561 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4621 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4681 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4741 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4801 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4861 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4921 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4981 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5041 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5101 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5161 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5221 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5281 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5341 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5401 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5461 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5521 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat
     5581 cccctagggt cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     5641 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     5701 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
     5761 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
     5821 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     5881 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     5941 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     6001 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
//