LOCUS       sequence-177995-        8873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the dCas9-MC 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 8873)
  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 8873)
  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 dCas9-MC (eGFP)
FEATURES             Location/Qualifiers
     source          1..8873
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        312..515
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     466..486
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             593..640
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             644..667
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5231..5251
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        5434..5840
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      5894..5903
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5900..5902,5903..5905,5906..6619)
                     /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(5900..5902,5903..5905,5906..6616)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5945..5966)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6206..6225)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6553..6574
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6753..6834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6790..6809)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        6884..6988
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6989..7057,7058..7849)
                     /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(7207..7226)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8020..8608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8509..8528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8684..8700
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     8685..8701
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(8743..8776)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        join(8805..8873,1..311)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
       61 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      121 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      181 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      241 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      301 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      361 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      421 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      481 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
      541 cttactggca tcccagtgtg gtggtacggg aattgacgtc ggcatgccag tgaagagacc
      601 tgctgccacc aagaaagctg gccaggccaa aaagaagaaa ctggactaca aagacgatga
      661 cgacaagacg cgtgataaga aatacagcat aggactggcc attggaacaa attctgtcgg
      721 ctgggcggtt attaccgatg aatataaggt tccgtccaag aaattcaagg ttctaggcaa
      781 tacagaccgc cacagtatta aaaagaattt gataggtgcc cttctgtttg acagtggcga
      841 gacagcggaa gcgaccagac tcaaacgaac agccaggaga cgttatacaa gacgaaagaa
      901 taggatctgt tatcttcaag aaatctttag caatgagatg gcgaaagtgg atgatagttt
      961 ctttcatcgc cttgaagagt cctttctggt agaagaagac aagaagcatg aaagacatcc
     1021 catctttggc aatatagtgg atgaagttgc ctatcatgag aaatatccaa ccatctacca
     1081 tctacgcaag aaactggtgg attccaccga taaagcggat ctgcgcctga tctatctagc
     1141 actagcgcac atgatcaagt ttagagggca ttttctgatc gagggcgatc tgaatcccga
     1201 taatagtgat gtagacaaac ttttcattca actggtgcag acttacaatc agctgtttga
     1261 agaaaacccc atcaacgcta gtggcgtgga tgccaaagcg atcctttccg ctcgcctgag
     1321 taaaagcaga cgcctggaaa atctcatcgc ccaactcccg ggggagaaga aaaatggact
     1381 gtttggtaac ctcatcgccc tgagcctggg gctgactccc aattttaaaa gcaattttga
     1441 tctggctgaa gatgccaaac tgcaacttag caaagatacc tacgatgatg atctggataa
     1501 tctgctggcg cagatcggcg atcagtatgc cgatctgttt ctggctgcca agaatctgag
     1561 cgatgccatc ctgcttagcg atattttgag agtgaatacc gaaataacca aggccccgtt
     1621 gagcgccagc atgatcaaac gctacgatga acatcatcag gacctgaccc ttctgaaagc
     1681 cctggttcgc cagcagcttc ctgaaaagta taaagagatt ttctttgatc agagcaagaa
     1741 cggctatgct gggtatatcg atggtggcgc ctctcaggaa gagttctata agttcattaa
     1801 accaatcctg gaaaagatgg atgggaccga ggaactgctg gtaaaattga atagggagga
     1861 tctgctacgc aagcagcgaa cttttgacaa cgggtccatc ccgcatcaga tccacctggg
     1921 ggagctacat gccatcctga gaagacagga agacttttat ccatttctga aagacaatcg
     1981 ggagaagatc gaaaagattc tgacctttcg catcccctat tatgttgggc ctctggcgag
     2041 aggaaacagt aggtttgctt ggatgacccg aaagtccgaa gaaacaatca ctccttggaa
     2101 cttcgaagaa gttgtcgata aaggggccag cgcccagagc tttatcgaac gcatgacaaa
     2161 ctttgataag aatcttccaa atgaaaaggt gcttcctaaa catagtctgc tttatgagta
     2221 ttttacggtt tataacgaac tgacaaaggt caaatatgtt accgaaggaa tgcgcaaacc
     2281 agcttttctt agcggggaac aaaagaaagc aatcgttgat ctgctcttca aaacaaatcg
     2341 caaagtgact gttaagcagc tgaaagaaga ttatttcaag aagatagaat gttttgatag
     2401 tgttgaaatc agcggcgttg aagatagatt caatgccagc ctggggactt accatgatct
     2461 gttgaaaatc atcaaggata aagacttcct ggataacgaa gaaaatgagg atattctgga
     2521 agatatcgtt ctgacactga ctctgtttga agatcgtgag atgatcgagg aaagacttaa
     2581 aacttatgcc cacctctttg atgataaggt aatgaaacaa cttaaaaggc gcagatatac
     2641 cgggtggggc agactgtccc gcaaactgat caatggaatc cgtgataagc agtccggaaa
     2701 gacaatactg gattttctga aaagcgatgg gtttgcaaat cgcaatttta tgcaactaat
     2761 tcatgatgat agtctgacat tcaaagaaga catccagaaa gctcaggtat ccggccaggg
     2821 agatagtctg catgaacata tcgctaatct ggccgggtct cccgccatca agaaaggaat
     2881 cctgcaaacc gtgaaagttg ttgatgaact ggtcaaagtg atgggtcgac ataagccaga
     2941 aaatattgtt atcgaaatgg ctagagaaaa ccaaacaacc cagaagggac aaaagaactc
     3001 gcgcgagagg atgaaacgca ttgaagaagg gattaaagaa ctgggcagtc aaatccttaa
     3061 agagcatccc gttgaaaata cccaactgca aaacgaaaag ctctatctct attatctcca
     3121 gaatggcaga gacatgtatg tagaccagga actggatatc aataggctga gtgattatga
     3181 tgtcgccgct atcgttccac agagtttcct taaagacgat agcatagaca ataaggtcct
     3241 gacgagatcc gataagaata gagggaaatc ggataacgtt cctagtgaag aagtggtcaa
     3301 gaagatgaag aactattgga gacagctttt gaacgcaaag ctgattaccc agaggaagtt
     3361 cgataatctg acgaaagccg aaagaggcgg gctgagtgaa cttgataaag ccgggtttat
     3421 taaacgccag ctggttgaaa cccgccagat taccaagcat gtagctcaga tcctggatag
     3481 tcgcatgaat accaaatacg atgaaaatga taaacttatc cgcgaggtta aagtaatcac
     3541 tctgaaatcc aaactggttt ccgacttccg caaagatttc cagttctata aagtgagaga
     3601 gatcaacaat taccatcatg cacatgatgc gtatttgaat gcagtcgttg gcaccgccct
     3661 gatcaagaag tatccaaaac ttgaatcgga gtttgtctat ggggattata aagtttatga
     3721 tgttaggaaa atgatcgcca agtccgagca ggaaatagga aaagctactg ctaagtattt
     3781 cttttactcc aatattatga acttcttcaa aacagaaatc acacttgcta atggcgagat
     3841 ccgcaaacgc cccttgattg aaaccaatgg tgaaaccggc gaaatcgtct gggataaagg
     3901 tcgcgatttt gcaacagtac gcaaagtgct gtcaatgccg caggtcaata tcgtcaagaa
     3961 aacagaagtg caaacaggag gcttctcaaa ggagagcatc ctgcctaaac ggaactcgga
     4021 caagcttatc gccaggaaga aagactggga cccaaagaaa tatggagggt ttgatagtcc
     4081 tacggtggcc tatagcgtcc ttgtagttgc caaggtagag aagggtaagt cgaagaagct
     4141 gaaatcagtt aaagagctgc ttggtattac aatcatggaa agaagttcat ttgaaaagaa
     4201 tccgatcgac tttctggaag ccaaaggcta taaggaagtt aagaaagacc tgattatcaa
     4261 acttcccaaa tatagtcttt tcgagctgga aaacgggagg aaacgaatgc tagccagtgc
     4321 aggcgaactg cagaaaggca atgagctagc cctaccatct aagtatgtaa acttcctgta
     4381 tctggccagt cattatgaaa agctgaaggg gagtccagaa gataacgaac agaagcagct
     4441 gtttgtagag caacataagc attatctgga tgagatcatc gagcagatta gtgaattttc
     4501 caagagggtt atcctggctg atgcaaatct ggataaagtt cttagtgctt ataacaaaca
     4561 tagagacaaa ccaataaggg aacaggctga aaatatcatc catctgttta cgctgacgaa
     4621 tcttggcgcc ccagccgcct tcaagtattt cgatacaaca atcgatagga aacgctatac
     4681 gtccacaaaa gaagttctgg atgcaaccct tattcatcag tcaattaccg ggctttatga
     4741 aacacgcatc gatctgagtc aacttggagg ggaccctgct tctggcggaa cttcatctga
     4801 tggtggcacg tcagacggag ggtcaagcaa cacaggcggt agctctgacg gagggagctc
     4861 agaaggcgaa cctgcgcatg cactgaagta caacctgact gagttcaaga agaccaaatc
     4921 aaacatcaac aaggcaagcc tcattgggta tagcaagttc aactcagaag gctacgtgta
     4981 cgaccctgag ttcacaggac caaccctgac tgcaagtgga gcaaactcac ggatcaagat
     5041 caaggacgga tctaacattc gcaagatgaa ctcagatgaa acgtttctgt acatgggctt
     5101 cgattcacaa gatggaaaga gggtgaacga gattgagttc ctgactgaaa accagaagat
     5161 cttcgtgtgc ggaaactcaa tctccgtgga agttcttgaa gcgatcatcg acaagattgg
     5221 agggaccgga ccgaagaaga aacggaaagt gtaatctaga tgatcagaac ggtcagcctg
     5281 ctacgtaaat tccgcatgcc actgattttc tgctatcttt cataataaag gcagccttgc
     5341 ctgaagtcgc aggttgactt catttttacc gggacgatct ctcgaagcgg atccatgaaa
     5401 gaccccacct gtaggtttgg caagctagct gcagtaacgc cattttgcaa ggcatggaaa
     5461 aataccaaac caagaataga gaagttcaga tcaagggcgg gtacatgaaa atagctaacg
     5521 ttgggccaaa caggatatct gcggtgagca gtttcggccc cggcccgggg ccaagaacag
     5581 atggtcaccg cagtttcggc cccggcccga ggccaagaac agatggtccc cagatatggc
     5641 ccaaccctca gcagtttctt aagacccatc agatgtttcc aggctccccc aaggacctga
     5701 aatgaccctg cgccttattt gaattaacca atcagcctgc ttctcgcttc tgttcgcgcg
     5761 cttctgcttc ccgagctcta taaaagagct cacaacccct cactcggcgc gccagtcctc
     5821 cgattgactg agtcgcccgg atgcatcttc atcggacagg ttagaccaga cttatccgga
     5881 taagtctggt cccgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     5941 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     6001 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     6061 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     6121 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     6181 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     6241 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     6301 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     6361 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     6421 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     6481 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     6541 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     6601 ggacgagctg tacaagtaac tagtacgtgc tacgagattt cgattccacc gccgccttct
     6661 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     6721 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     6781 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     6841 gttgtggttt gtcccgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     6901 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     6961 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     7021 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     7081 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     7141 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     7201 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     7261 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     7321 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     7381 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     7441 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     7501 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     7561 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     7621 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     7681 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     7741 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     7801 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     7861 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7921 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7981 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     8041 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     8101 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     8161 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     8221 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     8281 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     8341 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     8401 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     8461 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     8521 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     8581 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     8641 ggccttttgc tggccttttg ctcacatgtc tggtgaattc aagtcgaggt cgacggtatc
     8701 gataagctcg cttcacgaga ttccagcagg tcgagggacc taataacttc gtatagcata
     8761 cattatacga agttatatta agggttccaa gcttaagtcg cgttgacatt gattattgac
     8821 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tgg
//
