LOCUS       sequence-305808-        7434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeted m6A RNA methylation in mammalian cells.
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-M3nls
FEATURES             Location/Qualifiers
     source          1..7434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     246..265
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        246..264
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             314..334
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3317..3337
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4318..4335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4358..4375)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4364..4588
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4659..4675)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4659..4675)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4672..4694)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4683..4699
                     /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(4707..4713,4714..4731,4732..4737))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4752..4773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4890..4907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5061..5649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5141..5160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5820..6611,6612..6680))
                     /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     6443..6462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6681..6785)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6864..6883)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7055..7434
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gagatccgcg
      241 gccgctaata cgactcacta tagggagagc cgccaccatg aaacggacag ccgacggaag
      301 cgagttcgag tcaccaaaga agaagcggaa agtcaacatc cccgctctgg tggaaaacca
      361 gaagaagtac tttggcacct acagcgtgat ggccatgctg aacgctcaga ccgtgctgga
      421 ccacatccag aaggtggccg atattgaggg cgagcagaac gagaacaacg agaatctgtg
      481 gtttcacccc gtgatgagcc acctgtacaa cgccaagaac ggctacgaca agcagcccga
      541 gaaaaccatg ttcatcatcg agcggctgca gagctacttc ccattcctga agatcatggc
      601 cgagaaccag agagagtaca gcaacggcaa gtacaagcag aaccgcgtgg aagtgaacag
      661 caacgacatc ttcgaggtgc tgaagcgcgc cttcggcgtg ctgaagatgt acagggacct
      721 gaccaacgca tacaagacct acgaggaaaa gctgaacgac ggctgcgagt tcctgaccag
      781 cacagagcaa cctctgagcg gcatgatcaa caactactac acagtggccc tgcggaacat
      841 gaacgagaga tacggctaca agacagagga cctggccttc atccaggaca agcggttcaa
      901 gttcgtgaag gacgcctacg gcaagaaaaa gtcccaagtg aataccggat tcttcctgag
      961 cctgcaggac tacaacggcg acacacagaa gaagctgcac ctgagcggag tgggaatcgc
     1021 cctgctgatc tgcctgttcc tggacaagca gtacatcaac atctttctga gcaggctgcc
     1081 catcttctcc agctacaatg cccagagcga ggaacggcgg atcatcatca gatccttcgg
     1141 catcaacagc atcaagctgc ccaaggaccg gatccacagc gagaagtcca acaagagcgt
     1201 ggccatggat atgctcaacg aagtgaagcg gtgccccgac gagctgttca caacactgtc
     1261 tgccgagaag cagtcccggt tcagaatcat cagcgacgac cacaatgaag tgctgatgaa
     1321 gcggagcagc gacagattcg tgcctctgct gctgcagtat atcgattacg gcaagctgtt
     1381 cgaccacatc aggttccacg tgaacatggg caagctgaga tacctgctga aggccgacaa
     1441 gacctgcatc gacggccaga ccagagtcag agtgatcgag cagcccctga acggcttcgg
     1501 cagactggaa gaggccgaga caatgcggaa gcaagagaac ggcaccttcg gcaacagcgg
     1561 catccggatc agagacttcg agaacatgaa gcgggacgac gccaatcctg ccaactatcc
     1621 ctacatcgtg gacacctaca cacactacat cctggaaaac aacaaggtcg agatgtttat
     1681 caacgacaaa gaggacagcg ccccactgct gcccgtgatc gaggatgata gatacgtggt
     1741 caagacaatc cccagctgcc ggatgagcac cctggaaatt ccagccatgg ccttccacat
     1801 gtttctgttc ggcagcaaga aaaccgagaa gctgatcgtg gacgtgcaca accggtacaa
     1861 gagactgttc caggccatgc agaaagaaga agtgaccgcc gagaatatcg ccagcttcgg
     1921 aatcgccgag agcgacctgc ctcagaagat cctggatctg atcagcggca atgcccacgg
     1981 caaggatgtg gacgccttca tcagactgac cgtggacgac atgctgaccg acaccgagcg
     2041 gagaatcaag agattcaagg acgaccggaa gtccattcgg agcgccgaca acaagatggg
     2101 aaagagaggc ttcaagcaga tctccacagg caagctggcc gacttcctgg ccaaggacat
     2161 cgtgctgttt cagcccagcg tgaacgatgg cgagaacaag atcaccggcc tgaactaccg
     2221 gatcatgcag agcgccattg ccgtgtacga tagcggcgac gattacgagg ccaagcagca
     2281 gttcaagctg atgttcgaga aggcccggct gatcggcaag ggcacaacag agcctcatcc
     2341 atttctgtac aaggtgttcg cccgcagcat ccccgccaat gccgtcgagt tctacgagcg
     2401 ctacctgatc gagcggaagt tctacctgac cggcctgtcc aacgagatca agaaaggcaa
     2461 cagagtggat gtgcccttca tccggcggga ccagaacaag tggaaaacac ccgccatgaa
     2521 gaccctgggc agaatctaca gcgaggatct gcccgtggaa ctgcccagac agatgttcga
     2581 caatgagatc aagtcccacc tgaagtccct gccacagatg gaaggcatcg acttcaacaa
     2641 tgccaacgtg acctatctga tcgccgagta catgaagaga gtgctggacg acgacttcca
     2701 gaccttctac cagtggaacc gcaactaccg gtacatggac atgcttaagg gcgagtacga
     2761 cagaaagggc tccctgcagc actgcttcac cagcgtggaa gagagagaag gcctctggaa
     2821 agagcgggcc tccagaacag agcggtacag aaagcaggcc agcaacaaga tccgcagcaa
     2881 ccggcagatg agaaacgcca gcagcgaaga gatcgagaca atcctggata agcggctgag
     2941 caacagccgg aacgagtacc agaaaagcga gaaagtgatc cggcgctaca gagtgcagga
     3001 tgccctgctg tttctgctgg ccaaaaagac cctgaccgaa ctggccgatt tcgacggcga
     3061 gaggttcaaa ctgaaagaaa tcatgcccga cgccgagaag ggaatcctga gcgagatcat
     3121 gcccatgagc ttcaccttcg agaaaggcgg caagaagtac accatcacca gcgagggcat
     3181 gaagctgaag aactacggcg acttctttgt gctggctagc gacaagagga tcggcaacct
     3241 gctggaactc gtgggcagcg acatcgtgtc caaagaggat ggatccaaaa gaaccgccga
     3301 cggcagcgaa ttcgagccca agaagaagag gaaagtctct ggcagcgaga caccaggaac
     3361 aagcgagtca gcaacaccag agagccaaga gttctgtgac tatggcacca aagaggagtg
     3421 catgaaggct agcgacgctg atcgtccatg ccgtaagctg cacttccgtc gcatcattaa
     3481 caaacacacc gacgagagcc tgggcgactg cagcttcctg aacacctgct ttcacatgga
     3541 cacctgcaag tacgtgcact atgagatcga cgcgtgcatg gatagcgaag ctccgggcag
     3601 caaagatcac accccgagcc aggaactggc cctgacccag agcgtgggtg gtgacagcag
     3661 cgcggatcgt ctgttcccac cacagtggat ctgctgcgac attcgttacc tggatgtgag
     3721 catcctgggc aagtttgctg ttgtgatggc cgacccgccg tgggatattc acatggagct
     3781 gccgtatggt accctgaccg acgatgaaat gcgtcgcctg aacatcccgg tgctgcagga
     3841 cgatggcttc ctgtttctgt gggtgacggg tcgtgctatg gagctgggtc gtgaatgcct
     3901 gaacctgtgg ggttacgagc gtgtggacga aatcatttgg gtgaaaacca accagctgca
     3961 gcgtatcatt cgcaccggcc gtaccggtca ctggctgaac cacggcaagg agcactgcct
     4021 ggtgggcgtg aaaggcaacc cgcagggctt taaccagggt ctggactgcg atgtgatcgt
     4081 ggctgaagtg cgcagcacca gccacaagcc ggacgagatc tacggcatga ttgaacgcct
     4141 gagcccgggt acccgtaaaa ttgagctgtt cggccgtccg cacaacgtgc agccgaactg
     4201 gatcaccctg ggcaaccagc tggacggtat tcacctgctg gacccagatg tggtggctcg
     4261 ctttaaacag cgttatccgg atggcatcat tagcaaaccg aagaatctgt aaccggtcat
     4321 catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     4381 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4441 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4501 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     4561 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg
     4621 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4681 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4741 tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4801 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4861 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4921 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4981 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5041 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5101 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5161 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5221 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5281 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5341 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5401 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5461 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5521 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5581 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5641 ggatctcaag aagatccttt gatcttttct acggggtctg acactcagtg gaacgaaaac
     5701 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5761 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5821 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5881 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5941 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6001 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6061 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6121 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6181 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6241 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6301 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6361 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6421 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6481 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6541 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6601 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6661 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6721 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6781 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc
     6841 gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     6901 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     6961 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     7021 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
     7081 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     7141 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     7201 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     7261 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     7321 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     7381 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catg
//
