LOCUS       sequence-479364-        5605 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 5605)
  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 5605)
  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-SOD2 MTS-3xHA-TALE
            array-Nt.BspD6I(C)-P2A...
FEATURES             Location/Qualifiers
     source          1..5605
                     /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             520..546
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             550..576
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             580..606
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1849..1905
                     /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             join(1906..1908,1909..1911,1912..2625)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1906..1908,1909..1911,1912..2622)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1951..1972)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2212..2231)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2559..2580
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2961..2977)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2961..2977)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2974..2996)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2985..3001
                     /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(3009..3015,3016..3033,3034..3039))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3054..3075
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3192..3209)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3363..3951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3443..3462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4122..4913,4914..4982))
                     /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     4745..4764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4983..5087)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5166..5185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5357..5605,1..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 ccctgtcccg tgcggtttgt ggcacctccc gtcaactggc tccggttctg
      481 ggttatctgg gttcccgtca aaaacactcc ctgccggact acccgtatga tgttccggat
      541 tacgctggct acccatacga cgtcccagac tacgctggct acccatacga cgtcccagac
      601 tacgctatgg acatcgcgga cctcaggacg ctgggctaca gccagcaaca gcaagagaag
      661 atcaagccca aagtaaggag caccgtggcc cagcaccacg aggccctggt gggtcacggc
      721 ttcacccacg cgcatatcgt tgctctgagc caacatcccg cagctctggg taccgttgcg
      781 gtgaagtatc aggacatgat cgcggcactg cctgaagcta cacacgaagc catagtgggc
      841 gttggcaagc agtggagcgg tgccagagcg cttgaggcac tgttgacggt ggctggcgag
      901 ctgaggggac cgccactgca actggacacc ggccaactgc tgaagatcgc caagagggga
      961 ggcgtgacgg cggtggaggc cgtgcatgcc tggaggaacg cactgaccgg cgcgcccctg
     1021 aacagagacg ctggcgtctc tagcatcgtg gcccagctga gcagacccga ccctgccctc
     1081 gccgctctga ccaacgacca cctggtggcc ctggcttgcc tcgggggcag gccagctctt
     1141 gacgccgtga agaagggcct tggtggatcc cggcagctgg aggaggtgat tgatctgctg
     1201 gaggtgtacc atgagaagaa gaacgtgatc gaggagaaga tcaaggcccg gtttatcgcc
     1261 aacaagaaca ccgtgttcga gtggctgaca tggaacggct tcattattct gggcaatgcc
     1321 ctcgagtaca agaataattt cgtgatcgat gaagagctgc agcccgtgac acacgctgcc
     1381 ggcaaccagc ctgatatgga gattatctac gaggacttta ttgtgctggg cgaagtgacc
     1441 acatcaaagg gggccacaca gtttaagatg gagtctgagc cagtcactcg ccactacctg
     1501 aacaaaaaga aggagctgga aaaacagggc gtggagaagg aactgtattg cctgttcatc
     1561 gcccctgaaa tcaacaaaaa cacctttgag gagtttatga agtacaacat cgtgcagaac
     1621 accaggatca tccctctgtc tctgaagcag ttcaacatgc tgctgatggt gcagaagaag
     1681 ctgatcgaaa aaggccgcag actgtccagc tacgacatta agaacctgat ggtgagcctg
     1741 taccggacca caatcgagtg cgaaagaaag tacacccaga ttaaagccgg cctggaggaa
     1801 accctgaata actgggtggt ggacaaggag gtgagatttg gaagcggagc tactaacttc
     1861 agcctgctga agcaggctgg agatgtggag gagaaccctg gacctatggt gagcaagggc
     1921 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1981 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2041 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     2101 acctatggag tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     2161 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     2221 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2281 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2341 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     2401 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     2461 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     2521 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     2581 accgccgccg ggatcactct cggcatggac gagctgtaca agtagtctag aaccacgatc
     2641 gttatgctga tcatacccta atgatcccag caagataatg tcctgtcttc taagatgtgc
     2701 atcaagcctg gtacatactg aaaaccctat aaggtcctgg ataatttttg tttgattatt
     2761 cattgaagaa acatttattt tccaattgtg tgaagttttt gactgttaat aaaagaatct
     2821 gtcaaccatc aaaaccggtg tttaaacgaa gacaatagca ggcatgctgg ggatgcggtg
     2881 ggctctatgg cttctgaggc ggaaagaacc agctggggct cgataccgtc gacctctagc
     2941 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3001 attccacaca acatacgagc cggaagcata aagtgtaaag cctagggtgc ctaatgagtg
     3061 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3121 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3181 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3241 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3301 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3361 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3481 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3541 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3601 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3661 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3721 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3781 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3841 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3901 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3961 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4021 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4081 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4141 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4201 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4261 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4321 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4381 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4441 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4501 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4561 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4621 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4681 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4741 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4801 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4861 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4921 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4981 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5041 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5101 aaagtgccac ctgacgtcga cggatcggga gatcgatctc ccgatcccct agggtcgact
     5161 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     5221 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     5281 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     5341 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     5401 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     5461 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     5521 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     5581 cacttggcag tacatcaagt gtatc
//
