LOCUS       sequence-307627-        7434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeted m6A RNA methylation in mammalian cells, 
            methyltransferase-inactive mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7434)
  AUTHORS   Wilson C, Chen PJ, Miao Z, Liu DR
  TITLE     Programmable m(6)A modification of cellular RNAs with a 
            Cas13-directed methyltransferase.
  JOURNAL   Nat Biotechnol. 2020 Jun 29. pii: 10.1038/s41587-020-0572-6. doi: 
            10.1038/s41587-020-0572-6.
  PUBMED    32601430
REFERENCE   2  (bases 1 to 7434)
  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: "Nat 
            Biotechnol. 2020 Jun 29. pii: 10.1038/s41587-020-0572-6. doi: 
            10.1038/s41587-020-0572-6."
COMMENT     Sequence Label: pCMV-dCas13-inactive M3nls
FEATURES             Location/Qualifiers
     source          1..7434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     610..629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        610..628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             678..698
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3681..3701
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4682..4699
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4722..4739)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4728..4952
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5023..5039)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5023..5039)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5036..5058)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5047..5063
                     /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(5071..5077,5078..5095,5096..5101))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5116..5137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5254..5271)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5425..6013)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5505..5524)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6184..6975,6976..7044))
                     /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     6807..6826
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7045..7149)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7228..7247)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7419..7434,1..364)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc
      601 cgcggccgct aatacgactc actataggga gagccgccac catgaaacgg acagccgacg
      661 gaagcgagtt cgagtcacca aagaagaagc ggaaagtcaa catccccgct ctggtggaaa
      721 accagaagaa gtactttggc acctacagcg tgatggccat gctgaacgct cagaccgtgc
      781 tggaccacat ccagaaggtg gccgatattg agggcgagca gaacgagaac aacgagaatc
      841 tgtggtttca ccccgtgatg agccacctgt acaacgccaa gaacggctac gacaagcagc
      901 ccgagaaaac catgttcatc atcgagcggc tgcagagcta cttcccattc ctgaagatca
      961 tggccgagaa ccagagagag tacagcaacg gcaagtacaa gcagaaccgc gtggaagtga
     1021 acagcaacga catcttcgag gtgctgaagc gcgccttcgg cgtgctgaag atgtacaggg
     1081 acctgaccaa cgcatacaag acctacgagg aaaagctgaa cgacggctgc gagttcctga
     1141 ccagcacaga gcaacctctg agcggcatga tcaacaacta ctacacagtg gccctgcgga
     1201 acatgaacga gagatacggc tacaagacag aggacctggc cttcatccag gacaagcggt
     1261 tcaagttcgt gaaggacgcc tacggcaaga aaaagtccca agtgaatacc ggattcttcc
     1321 tgagcctgca ggactacaac ggcgacacac agaagaagct gcacctgagc ggagtgggaa
     1381 tcgccctgct gatctgcctg ttcctggaca agcagtacat caacatcttt ctgagcaggc
     1441 tgcccatctt ctccagctac aatgcccaga gcgaggaacg gcggatcatc atcagatcct
     1501 tcggcatcaa cagcatcaag ctgcccaagg accggatcca cagcgagaag tccaacaaga
     1561 gcgtggccat ggatatgctc aacgaagtga agcggtgccc cgacgagctg ttcacaacac
     1621 tgtctgccga gaagcagtcc cggttcagaa tcatcagcga cgaccacaat gaagtgctga
     1681 tgaagcggag cagcgacaga ttcgtgcctc tgctgctgca gtatatcgat tacggcaagc
     1741 tgttcgacca catcaggttc cacgtgaaca tgggcaagct gagatacctg ctgaaggccg
     1801 acaagacctg catcgacggc cagaccagag tcagagtgat cgagcagccc ctgaacggct
     1861 tcggcagact ggaagaggcc gagacaatgc ggaagcaaga gaacggcacc ttcggcaaca
     1921 gcggcatccg gatcagagac ttcgagaaca tgaagcggga cgacgccaat cctgccaact
     1981 atccctacat cgtggacacc tacacacact acatcctgga aaacaacaag gtcgagatgt
     2041 ttatcaacga caaagaggac agcgccccac tgctgcccgt gatcgaggat gatagatacg
     2101 tggtcaagac aatccccagc tgccggatga gcaccctgga aattccagcc atggccttcc
     2161 acatgtttct gttcggcagc aagaaaaccg agaagctgat cgtggacgtg cacaaccggt
     2221 acaagagact gttccaggcc atgcagaaag aagaagtgac cgccgagaat atcgccagct
     2281 tcggaatcgc cgagagcgac ctgcctcaga agatcctgga tctgatcagc ggcaatgccc
     2341 acggcaagga tgtggacgcc ttcatcagac tgaccgtgga cgacatgctg accgacaccg
     2401 agcggagaat caagagattc aaggacgacc ggaagtccat tcggagcgcc gacaacaaga
     2461 tgggaaagag aggcttcaag cagatctcca caggcaagct ggccgacttc ctggccaagg
     2521 acatcgtgct gtttcagccc agcgtgaacg atggcgagaa caagatcacc ggcctgaact
     2581 accggatcat gcagagcgcc attgccgtgt acgatagcgg cgacgattac gaggccaagc
     2641 agcagttcaa gctgatgttc gagaaggccc ggctgatcgg caagggcaca acagagcctc
     2701 atccatttct gtacaaggtg ttcgcccgca gcatccccgc caatgccgtc gagttctacg
     2761 agcgctacct gatcgagcgg aagttctacc tgaccggcct gtccaacgag atcaagaaag
     2821 gcaacagagt ggatgtgccc ttcatccggc gggaccagaa caagtggaaa acacccgcca
     2881 tgaagaccct gggcagaatc tacagcgagg atctgcccgt ggaactgccc agacagatgt
     2941 tcgacaatga gatcaagtcc cacctgaagt ccctgccaca gatggaaggc atcgacttca
     3001 acaatgccaa cgtgacctat ctgatcgccg agtacatgaa gagagtgctg gacgacgact
     3061 tccagacctt ctaccagtgg aaccgcaact accggtacat ggacatgctt aagggcgagt
     3121 acgacagaaa gggctccctg cagcactgct tcaccagcgt ggaagagaga gaaggcctct
     3181 ggaaagagcg ggcctccaga acagagcggt acagaaagca ggccagcaac aagatccgca
     3241 gcaaccggca gatgagaaac gccagcagcg aagagatcga gacaatcctg gataagcggc
     3301 tgagcaacag ccggaacgag taccagaaaa gcgagaaagt gatccggcgc tacagagtgc
     3361 aggatgccct gctgtttctg ctggccaaaa agaccctgac cgaactggcc gatttcgacg
     3421 gcgagaggtt caaactgaaa gaaatcatgc ccgacgccga gaagggaatc ctgagcgaga
     3481 tcatgcccat gagcttcacc ttcgagaaag gcggcaagaa gtacaccatc accagcgagg
     3541 gcatgaagct gaagaactac ggcgacttct ttgtgctggc tagcgacaag aggatcggca
     3601 acctgctgga actcgtgggc agcgacatcg tgtccaaaga ggatggatcc aaaagaaccg
     3661 ccgacggcag cgaattcgag cccaagaaga agaggaaagt ctctggcagc gagacaccag
     3721 gaacaagcga gtcagcaaca ccagagagcc aagagttctg tgactatggc accaaagagg
     3781 agtgcatgaa ggctagcgac gctgatcgtc catgccgtaa gctgcacttc cgtcgcatca
     3841 ttaacaaaca caccgacgag agcctgggcg actgcagctt cctgaacacc tgctttcaca
     3901 tggacacctg caagtacgtg cactatgaga tcgacgcgtg catggatagc gaagctccgg
     3961 gcagcaaaga tcacaccccg agccaggaac tggccctgac ccagagcgtg ggtggtgaca
     4021 gcagcgcgga tcgtctgttc ccaccacagt ggatctgctg cgacattcgt tacctggatg
     4081 tgagcatcct gggcaagttt gctgttgtga tggccgcccc gccgtgggat attcacatgg
     4141 agctgccgta tggtaccctg accgacgatg aaatgcgtcg cctgaacatc ccggtgctgc
     4201 aggacgatgg cttcctgttt ctgtgggtga cgggtcgtgc tatggagctg ggtcgtgaat
     4261 gcctgaacct gtggggttac gagcgtgtgg acgaaatcat ttgggtgaaa accaaccagc
     4321 tgcagcgtat cattcgcacc ggccgtaccg gtcactggct gaaccacggc aaggagcact
     4381 gcctggtggg cgtgaaaggc aacccgcagg gctttaacca gggtctggac tgcgatgtga
     4441 tcgtggctga agtgcgcagc accagccaca agccggacga gatctacggc atgattgaac
     4501 gcctgagccc gggtacccgt aaaattgagc tgttcggccg tccgcacaac gtgcagccga
     4561 actggatcac cctgggcaac cagctggacg gtattcacct gctggaccca gatgtggtgg
     4621 ctcgctttaa acagcgttat ccggatggca tcattagcaa accgaagaat ctgtaaccgg
     4681 tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag
     4741 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4801 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     4861 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     4921 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     4981 ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5041 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5101 agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5161 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5221 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5281 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5341 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5401 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5461 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5521 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5581 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5641 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5701 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5761 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5821 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5881 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5941 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6001 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc agtggaacga
     6061 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6121 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6181 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6241 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6301 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6361 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6421 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6481 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6541 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6601 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6661 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6721 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6781 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6841 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6901 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6961 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7021 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7081 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7141 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc
     7201 tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca
     7261 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     7321 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     7381 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgac
//
