LOCUS       sequence-177997-        5023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the MN fragment only of the sMTase system for targeted DNA
            methylation in mammalian cells. Contains a eGFP marker expressed off
            separate promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5023)
  AUTHORS   Xiong T, Meister GE, Workman RE, Kato NC, Spellberg MJ, Turker F, 
            Timp W, Ostermeier M, Novina CD
  TITLE     Targeted DNA methylation in human cells using engineered 
            dCas9-methyltransferases.
  JOURNAL   Sci Rep. 2017 Jul 27;7(1):6732. doi: 10.1038/s41598-017-06757-0.
  PUBMED    28751638
REFERENCE   2  (bases 1 to 5023)
  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"; journalName: "Sci Rep."; 
            date: "2017-07-27"; volume: "7(1):6732. doi"; pages: " 
            10.1038/s41598-017-06757-0"
COMMENT     Sequence Label: pCMV MN (eGFP)
FEATURES             Location/Qualifiers
     source          1..5023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..39
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     24..40
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(82..115)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        144..523
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        524..727
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     678..698
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             799..819
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             820..846
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        1796..2202
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2256..2265
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2262..2264,2265..2267,2268..2981)
                     /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(2262..2264,2265..2267,2268..2978)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2307..2328)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2568..2587)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2915..2936
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3115..3196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3152..3171)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3246..3350
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3351..3419,3420..4211)
                     /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(3569..3588)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4382..4970
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4871..4890
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacatgtct ggtgaattca agtcgaggtc gacggtatcg ataagctcgc ttcacgagat
       61 tccagcaggt cgagggacct aataacttcg tatagcatac attatacgaa gttatattaa
      121 gggttccaag cttaagtcgc gttgacattg attattgact agttattaat agtaatcaat
      181 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      241 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      301 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      361 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      421 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      481 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      541 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      601 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      661 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      721 cagagctctc tggctaacta gagaacccac tgcttactgg catcccagtg tggtggtacg
      781 ggaattgacg tcggcatgcc gaagaagaaa cggaaagtgt acccatacga cgttccagac
      841 tacgctagta aggtggagaa caaaacaaag aaactccggg tgttcgaggc ttttgctggg
      901 atcggggccc aacggaaggc cttggagaaa gtaaggaagg acgagtatga gatcgttggg
      961 cttgctgagt ggtatgtccc tgccattgtg atgtatcaag cgatccacaa taacttccat
     1021 actaagcttg agtacaagtc cgtgtctagg gaagagatga ttgattactt ggagaacaag
     1081 actctatctt ggaatagcaa gaaccccgtg agcaatggct actggaagag gaagaaggat
     1141 gatgaactaa agattatcta caacgctata aagctctcag aaaaggaggg gaatatcttc
     1201 gacatcaggg atctatataa gcggactctc aaaaacattg accttctgac atacagcttc
     1261 ccttgccagg atctgtccca gcaaggcatt cagaagggta tgaaaagagg atctggcact
     1321 aggtctggac tcctgtggga gattgaaagg gcactggact caactgagaa gaacgatctt
     1381 ccaaagtatc tattgatgga gaacgttggc gctttactcc ataagaagaa cgaggaagag
     1441 ctaaaccagt ggaaacagaa acttgagtct ttgggatacc aaaactcaat tgaggtgttg
     1501 aacgctgccg attttggctc ctctcaggct agaaggagag tgttcatgat aagcactctt
     1561 aacgagttcg ttgaactgcc taaaggcgac aagaagccta agtccatcaa gaaagttctg
     1621 aacaagatcg tgtcagagaa ggacattctg aacaacctgt aagctgattt tctgctatct
     1681 ttcataataa aggcagcctt gcctgaagtc gcaggttgac ttcattttta ccgggacgat
     1741 ctctcgaagc ggatccatga aagaccccac ctgtaggttt ggcaagctag ctgcagtaac
     1801 gccattttgc aaggcatgga aaaataccaa accaagaata gagaagttca gatcaagggc
     1861 gggtacatga aaatagctaa cgttgggcca aacaggatat ctgcggtgag cagtttcggc
     1921 cccggcccgg ggccaagaac agatggtcac cgcagtttcg gccccggccc gaggccaaga
     1981 acagatggtc cccagatatg gcccaaccct cagcagtttc ttaagaccca tcagatgttt
     2041 ccaggctccc ccaaggacct gaaatgaccc tgcgccttat ttgaattaac caatcagcct
     2101 gcttctcgct tctgttcgcg cgcttctgct tcccgagctc tataaaagag ctcacaaccc
     2161 ctcactcggc gcgccagtcc tccgattgac tgagtcgccc ggatgcatct tcatcggaca
     2221 ggttagatca gacttatccg gataagtctg gtcccgccac catggtgagc aagggcgagg
     2281 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2341 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2401 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     2461 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2521 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2581 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2641 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2701 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     2761 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     2821 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     2881 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2941 ccgccgggat cactctcggc atggacgagc tgtacaagta actagtacgt gctacgagat
     3001 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3061 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     3121 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3181 gcattttttt cactgcattc tagttgtggt ttgtcccgtc aggtggcact tttcggggaa
     3241 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3301 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3361 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3421 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3481 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3541 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3601 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3661 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3721 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3781 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3841 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3901 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3961 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4021 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
     4081 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4141 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4201 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4261 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4321 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4381 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4441 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4501 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4561 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4621 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4681 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4741 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4801 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4861 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4921 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4981 acgcggcctt tttacggttc ctggcctttt gctggccttt tgc
//
