LOCUS       sequence-479363-        5347 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Plasmids for constructing mitoBEs v2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5347)
  AUTHORS   Zhang X, Zhang X, Ren J, Li J, Wei X, Yu Y, Yi Z, Wei W
  TITLE     Precise modelling of mitochondrial diseases using optimized mitoBEs.
  JOURNAL   Nature. 2025 Jan 22. doi: 10.1038/s41586-024-08469-8.
  PUBMED    39843744
REFERENCE   2  (bases 1 to 5347)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2025 Jan 22. doi: 10.1038/s41586-024-08469-8."
COMMENT     Sequence Label: pCMV-COX8 MTS-3xFlag-TALE array-TadA8e(V106W,
            ...
FEATURES             Location/Qualifiers
     source          1..5347
                     /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"
     CDS             1654..1710
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1711..2421
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1856..1874)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2124..2143
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2703..2719)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2703..2719)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2716..2738)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2727..2743
                     /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(2751..2757,2758..2775,2776..2781))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2796..2817
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2934..2951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3105..3693)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3185..3204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3864..4655,4656..4724))
                     /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     4487..4506
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4725..4829)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4908..4927)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5099..5347,1..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 cccctgaaca gagacgctgg cgtctctagc
     1021 atcgtggccc agctgagcag acccgaccct gccctcgccg ctctgaccaa cgaccacctg
     1081 gtggccctgg cttgcctcgg gggcaggcca gctcttgacg ccgtgaagaa gggccttggt
     1141 ggatcctctg aggtggagtt ttcccacgag tactggatga gacatgccct gaccctggcc
     1201 aagagggcac gcgatgagag ggagttccct gtgggagccg tgctggtgct gaacaataga
     1261 gtgatcggcg agggctggaa cagagccatc ggcctgcacg acccaacagc ccatgccgaa
     1321 attatggccc tgagacaggg cggcctggtc atgcagaact acagactgat tgacgccacc
     1381 ctgtacgtga cattcgagcc ttgcgtgatg tgcgccggcg ccatgatcca ctctaggatc
     1441 ggccgcgtgg tgtttggctg gaggaactcc aaaaggggcg ccgcaggctc cctgatgaac
     1501 gtgctgaatt accccggcat gaatcaccgc gtcgaaatta ccgagggaat cctggcagat
     1561 gaatgtgccg ccctgctgtg cgatttctac cggatgccta gacaggtgtt caatgctcag
     1621 aagaaggccc agagctccat caatggaagc ggagctacta acttcagcct gctgaagcaa
     1681 gctggagatg tggaggagaa ccctggacct atggtgagca agggcgagga ggataacatg
     1741 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     1801 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     1861 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     1921 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     1981 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     2041 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     2101 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     2161 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     2221 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     2281 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac
     2341 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     2401 atggacgagc tgtacaagtg atctagaggg gtctttgtcc tctgtactgt ctctctcctt
     2461 gcccctaacc caaaaagctt catttttctg tgtaggctgc acaagagcct tgattgaaga
     2521 tatattcttt ctgaacagta tttaaggttt ccaataaaat gtacacccct cagaaaccgg
     2581 tgtttaaacg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2641 gcggaaagaa ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca
     2701 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2761 gccggaagca taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt
     2821 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2881 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2941 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3001 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3061 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3121 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3181 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3241 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3301 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3361 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3421 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3481 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3541 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3601 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3661 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3721 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3781 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3841 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3901 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3961 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4021 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4081 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4141 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4201 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4261 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4321 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4381 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4441 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4501 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4561 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4621 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4681 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4741 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4801 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4861 gacggatcgg gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct
     4921 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     4981 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     5041 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
     5101 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     5161 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     5221 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     5281 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     5341 gtgtatc
//
