LOCUS       sequence-361822-        5836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP tagged methyl-CpG recognition motif targeted to 
            mitochondria in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5836)
  AUTHORS   Zhao H, Ma D, Xie J, Sanchez O, Huang F, Yuan C
  TITLE     Live-Cell Probe for In Situ Single-Cell Monitoring of Mitochondrial 
            DNA Methylation.
  JOURNAL   ACS Sens. 2021 Oct 22;6(10):3575-3586. doi: 
            10.1021/acssensors.1c00731. Epub 2021 Sep 29.
  PUBMED    34586782
REFERENCE   2  (bases 1 to 5836)
  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: 
            "10.1021/acssensors.1c00731"; journalName: "ACS Sens"; date: 
            "2021-10-22- 22"; volume: "6"; issue: "10"; pages: "3575-3586"
COMMENT     Sequence Label: Mito-meCpG
FEATURES             Location/Qualifiers
     source          1..5836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1327..1336
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1333..1335,1336..1338,1339..2052)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1378..1399)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1639..1658)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1986..2007
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2081..2090
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2094..2228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2131..2150)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2185..2204
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2301..2658
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2301..2492
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2324..2344)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2509..2644
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2678..2695)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2678..2694)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2687..2709)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2907..3362
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2994..3013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3204..3225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3379..3398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3533..3555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3608..3626)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3694..3798
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3799..3867,3868..4659)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRDDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4017..4036)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4830..5418
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5319..5338
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5572..5589
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5742..5747,5748..5765,5766..5772)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5780..5796
                     /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)."
     primer_bind     5785..5807
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5804..5820
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5804..5820
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatctg cctggagacg ccatccacgc tgttttgacc tccatagaag
      661 acaccgggac cgatccagcc tccgcggccc gggatgtcag tcctgacacc tctgctgctg
      721 cgaggactga ctggaagcgc caggcgcctg cctggtagcg gcagcggtag cggttccggg
      781 atgtcagtcc tgacacctct gctgctgcga ggactgactg gaagcgccag gcgcctgcct
      841 ggtagcggca gcggtagcgg ttccggaggc agcgaattcg ctgaggactg gctggacagc
      901 ccggccctgg gccctggctg gaagcgccgc gaagtctttc gcaagtcagg ggccaccgcg
      961 ggacgctcag acacctatta ccagagcccc acaggagaca ggatccgaag caaagttgag
     1021 ctgactcgat acctgggccc tgcgggcgat ctcaccctct tcgacttcaa acaaggcatc
     1081 ttgggcggtg gccaattcgc tgaggactgg ctggacagcc cggccctggg ccctggctgg
     1141 aagcgccgcg aagtctttcg caagtcaggg gccaccgcgg gacgctcaga cacctattac
     1201 cagagcccca caggagacag gatccgaagc aaagttgagc tgactcgata cctgggccct
     1261 gcgggcgatc tcaccctctt cgacttcaaa caaggcatct tgggcggtgg ccaattcgta
     1321 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1381 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1441 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1501 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1561 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1621 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1681 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1741 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1801 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1861 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1921 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     1981 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2041 ctgtacaagt aatcctctag agtcgacctg cagaagcttg gccgccatgg cccaacttgt
     2101 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2161 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     2221 tctggatcga tcgggaatta attcggcgca gcaccatggc ctgaaataac ctctgaaaga
     2281 ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     2341 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2401 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2461 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     2521 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     2581 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     2641 ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc
     2701 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccccttcg
     2761 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgtagcc
     2821 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2881 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     2941 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3001 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3061 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3121 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3181 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3241 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3301 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     3361 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc aactccgcta
     3421 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3481 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3541 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagta ttcttgaaga
     3601 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     3661 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3721 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3781 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3841 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     3901 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3961 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     4021 tgtggcgcgg tattatcccg tgatgacgcc gggcaagagc aactcggtcg ccgcatacac
     4081 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     4141 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     4201 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     4261 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     4321 gagcgtgaca ccacgatgcc agcagcaatg gcaacaacgt tgcgcaaact attaactggc
     4381 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     4441 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     4501 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     4561 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4621 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4681 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4741 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4801 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4861 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4921 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     4981 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5041 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5101 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5161 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5221 cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5281 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5341 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5401 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5461 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5521 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     5581 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     5641 attcattaat ccagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     5701 gcaattaatg tgagttacct cactcattag gcaccccagg ctttacactt tatgcttccg
     5761 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     5821 catgattacg aattaa
//
