LOCUS       sequence-485967-        5614 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Plasmids for constructing mitoBEs v2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5614)
  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 5614)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 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-CBE6d-UGI-P2A-...
FEATURES             Location/Qualifiers
     source          1..5614
                     /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             1663..1911
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             1921..1977
                     /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             1978..2688
                     /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(2123..2141)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2391..2410
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2970..2986)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2970..2986)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2983..3005)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2994..3010
                     /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(3018..3024,3025..3042,3043..3048))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3063..3084
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3201..3218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3372..3960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3452..3471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4131..4922,4923..4991))
                     /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     4754..4773
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4992..5096)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5175..5194)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5366..5614,1..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 ggatccagtt ctgaggtgga gttttcccac gagtactgga tgagacatgc cctgaccctg
     1201 gccaagaggg cacgggatga gggagaggcg cctgtgggag ccgtgctggt gctgaacaat
     1261 agagtgatcg gcgagggctg gtgccgtcgt atcggcctgc acgacccaac agcccatgcc
     1321 gaaattatgg ccctgagaca gggcggcctg gtcatgcaga acccaagact gattgacgcc
     1381 accctgtacg tgacattcga gccttgcgtg atgtgcgccg gcgccatgat caactctagg
     1441 atcggccgcg tggtgtttgg cgtgaggaac tcaaaaagag gcgccgcagg ctccctgatg
     1501 aacgtgctga actaccccgg catgaatcac cgcgtcgaaa ttaccgaggg aatcctggca
     1561 gatgaatgtg ccgccctgct gtgcgatttc tatcggatgc ctagacaggt gttcaatgct
     1621 cagaagaagg cccagagctc catcaacggg agcgggggga gcaccaacct gtctgacatc
     1681 atcgagaagg agacaggcaa gcagctggtc atccaggaga gcatcctgat gctgcccgaa
     1741 gaagtcgaag aagtgatcgg aaacaagcct gagagcgata tcctggtcca taccgcctac
     1801 gacgagagta ccgacgaaaa tgtgatgctg ctgacatccg acgcaccaga gtataagccc
     1861 tgggctctgg tcatccagga ttccaacgga gagaacaaaa tcaaaatgct gggaagcgga
     1921 gctactaact tcagcctgct gaagcaagct ggagatgtgg aggagaaccc tggacctatg
     1981 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     2041 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     2101 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     2161 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     2221 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     2281 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     2341 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     2401 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     2461 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     2521 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     2581 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     2641 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtgatc tagaggggtc
     2701 tttgtcctct gtactgtctc tctccttgcc cctaacccaa aaagcttcat ttttctgtgt
     2761 aggctgcaca agagccttga ttgaagatat attctttctg aacagtattt aaggtttcca
     2821 ataaaatgta cacccctcag aaaccggtgt ttaaacgaag acaatagcag gcatgctggg
     2881 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg
     2941 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3001 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc
     3061 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3121 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3181 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3241 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3301 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3361 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3421 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3481 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3541 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3601 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3661 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc accactggca
     3721 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3781 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3841 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3901 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3961 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4021 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4081 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4141 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4201 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4261 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4321 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4381 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4441 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4501 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4561 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4621 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4681 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4741 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4801 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4861 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4921 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4981 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5041 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5101 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta
     5161 gggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     5221 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     5281 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     5341 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     5401 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     5461 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     5521 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     5581 taaactgccc acttggcagt acatcaagtg tatc
//
