LOCUS       sequence-371211-        5781 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 5781)
  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 5781)
  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..5781
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      443..452
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(449..451,452..454,455..1168)
                     /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(494..515)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(755..774)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1102..1123
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1197..1206
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1210..1344
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1247..1266)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1301..1320
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1413..1770
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1413..1604
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1436..1456)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1621..1756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1790..1807)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1790..1806)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1799..1821)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2019..2474
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2106..2125)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2316..2337
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2491..2510)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2610..2632
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2670..2688)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2756..2860
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2861..2929,2930..3721)
                     /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     complement(3079..3098)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3892..4480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4381..4400
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4634..4651
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4768..4789
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4804..4809,4810..4827,4828..4834)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4842..4858
                     /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     4847..4869
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4866..4882
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4866..4882
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        4911..5290
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5291..5494
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5445..5465
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5491..5515
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 ggactggctg gacagcccgg ccctgggccc tggctggaag cgccgcgaag tctttcgcaa
       61 gtcaggggcc accgcgggac gctcagacac ctattaccag agccccacag gagacaggat
      121 ccgaagcaaa gttgagctga ctcgatacct gggccctgcg ggcgatctca ccctcttcga
      181 cttcaaacaa ggcatcttgg gcggtggcca attcgctgag gactggctgg acagcccggc
      241 cctgggccct ggctggaagc gccgcgaagt ctttcgcaag tcaggggcca ccgcgggacg
      301 ctcagacacc tattaccaga gccccacagg agacaggatc cgaagcaaag ttgagctgac
      361 tcgatacctg ggccctgcgg gcgatctcac cctcttcgac ttcaaacaag gcatcttggg
      421 cggtggccaa ttcgtaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
      481 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      541 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      601 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
      661 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      721 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
      781 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
      841 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
      901 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
      961 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     1021 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga
     1081 cccaaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1141 tctcggcatg gacgagctgt acaagtaatc ctctagagtc gacctgcaga agcttggccg
     1201 ccatggccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     1261 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     1321 aatgtatctt atcatgtctg gatcgggaat taattcggcg cagcaccatg gcctgaaata
     1381 acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat
     1441 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     1501 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     1561 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     1621 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     1681 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     1741 ggcttttttg gaggcctagg cttttgcaaa aagctgttaa cagcttggca ctggccgtcg
     1801 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     1861 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     1921 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     1981 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     2041 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2101 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2161 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2221 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2281 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2341 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc
     2401 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2461 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2521 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2581 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     2641 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     2701 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     2761 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2821 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2881 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2941 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3001 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3061 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3121 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3181 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3241 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3301 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3361 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3421 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3481 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3541 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3601 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3661 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3721 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3781 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3841 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3901 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3961 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4021 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4081 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4141 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4201 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4261 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4321 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4381 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4441 atttttgtga tgctcgtcag gggggcagag cctatggaaa aacgccagca acgcggcctt
     4501 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4561 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4621 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     4681 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     4741 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     4801 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     4861 tcacacagga aacagctatg acatgattac gaattaattc gagctcgccc gacattgatt
     4921 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     4981 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     5041 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     5101 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     5161 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     5221 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     5281 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     5341 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     5401 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     5461 gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatctgcct
     5521 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc
     5581 gcggcccggg atgtcagtcc tgacacctct gctgctgcga ggactgactg gaagcgccag
     5641 gcgcctgcct ggtagcggca gcggtagcgg ttccgggatg tcagtcctga cacctctgct
     5701 gctgcgagga ctgactggaa gcgccaggcg cctgcctggt agcggcagcg gtagcggttc
     5761 cggaggcagc gaattcgctg a
//
