LOCUS       sequence-305821-        8280 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 8280)
  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 8280)
  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-M3M14nes
FEATURES             Location/Qualifiers
     source          1..8280
                     /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             3239..3265
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             5164..5181
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5204..5221)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5210..5434
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5505..5521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5505..5521)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5518..5540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5529..5545
                     /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(5553..5559,5560..5577,5578..5583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5598..5619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5736..5753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5907..6495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5987..6006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6666..7457,7458..7526))
                     /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     7289..7308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7527..7631)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7710..7729)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7901..8280
                     /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 aacatccccg ctctggtgga
      301 aaaccagaag aagtactttg gcacctacag cgtgatggcc atgctgaacg ctcagaccgt
      361 gctggaccac atccagaagg tggccgatat tgagggcgag cagaacgaga acaacgagaa
      421 tctgtggttt caccccgtga tgagccacct gtacaacgcc aagaacggct acgacaagca
      481 gcccgagaaa accatgttca tcatcgagcg gctgcagagc tacttcccat tcctgaagat
      541 catggccgag aaccagagag agtacagcaa cggcaagtac aagcagaacc gcgtggaagt
      601 gaacagcaac gacatcttcg aggtgctgaa gcgcgccttc ggcgtgctga agatgtacag
      661 ggacctgacc aacgcataca agacctacga ggaaaagctg aacgacggct gcgagttcct
      721 gaccagcaca gagcaacctc tgagcggcat gatcaacaac tactacacag tggccctgcg
      781 gaacatgaac gagagatacg gctacaagac agaggacctg gccttcatcc aggacaagcg
      841 gttcaagttc gtgaaggacg cctacggcaa gaaaaagtcc caagtgaata ccggattctt
      901 cctgagcctg caggactaca acggcgacac acagaagaag ctgcacctga gcggagtggg
      961 aatcgccctg ctgatctgcc tgttcctgga caagcagtac atcaacatct ttctgagcag
     1021 gctgcccatc ttctccagct acaatgccca gagcgaggaa cggcggatca tcatcagatc
     1081 cttcggcatc aacagcatca agctgcccaa ggaccggatc cacagcgaga agtccaacaa
     1141 gagcgtggcc atggatatgc tcaacgaagt gaagcggtgc cccgacgagc tgttcacaac
     1201 actgtctgcc gagaagcagt cccggttcag aatcatcagc gacgaccaca atgaagtgct
     1261 gatgaagcgg agcagcgaca gattcgtgcc tctgctgctg cagtatatcg attacggcaa
     1321 gctgttcgac cacatcaggt tccacgtgaa catgggcaag ctgagatacc tgctgaaggc
     1381 cgacaagacc tgcatcgacg gccagaccag agtcagagtg atcgagcagc ccctgaacgg
     1441 cttcggcaga ctggaagagg ccgagacaat gcggaagcaa gagaacggca ccttcggcaa
     1501 cagcggcatc cggatcagag acttcgagaa catgaagcgg gacgacgcca atcctgccaa
     1561 ctatccctac atcgtggaca cctacacaca ctacatcctg gaaaacaaca aggtcgagat
     1621 gtttatcaac gacaaagagg acagcgcccc actgctgccc gtgatcgagg atgatagata
     1681 cgtggtcaag acaatcccca gctgccggat gagcaccctg gaaattccag ccatggcctt
     1741 ccacatgttt ctgttcggca gcaagaaaac cgagaagctg atcgtggacg tgcacaaccg
     1801 gtacaagaga ctgttccagg ccatgcagaa agaagaagtg accgccgaga atatcgccag
     1861 cttcggaatc gccgagagcg acctgcctca gaagatcctg gatctgatca gcggcaatgc
     1921 ccacggcaag gatgtggacg ccttcatcag actgaccgtg gacgacatgc tgaccgacac
     1981 cgagcggaga atcaagagat tcaaggacga ccggaagtcc attcggagcg ccgacaacaa
     2041 gatgggaaag agaggcttca agcagatctc cacaggcaag ctggccgact tcctggccaa
     2101 ggacatcgtg ctgtttcagc ccagcgtgaa cgatggcgag aacaagatca ccggcctgaa
     2161 ctaccggatc atgcagagcg ccattgccgt gtacgatagc ggcgacgatt acgaggccaa
     2221 gcagcagttc aagctgatgt tcgagaaggc ccggctgatc ggcaagggca caacagagcc
     2281 tcatccattt ctgtacaagg tgttcgcccg cagcatcccc gccaatgccg tcgagttcta
     2341 cgagcgctac ctgatcgagc ggaagttcta cctgaccggc ctgtccaacg agatcaagaa
     2401 aggcaacaga gtggatgtgc ccttcatccg gcgggaccag aacaagtgga aaacacccgc
     2461 catgaagacc ctgggcagaa tctacagcga ggatctgccc gtggaactgc ccagacagat
     2521 gttcgacaat gagatcaagt cccacctgaa gtccctgcca cagatggaag gcatcgactt
     2581 caacaatgcc aacgtgacct atctgatcgc cgagtacatg aagagagtgc tggacgacga
     2641 cttccagacc ttctaccagt ggaaccgcaa ctaccggtac atggacatgc ttaagggcga
     2701 gtacgacaga aagggctccc tgcagcactg cttcaccagc gtggaagaga gagaaggcct
     2761 ctggaaagag cgggcctcca gaacagagcg gtacagaaag caggccagca acaagatccg
     2821 cagcaaccgg cagatgagaa acgccagcag cgaagagatc gagacaatcc tggataagcg
     2881 gctgagcaac agccggaacg agtaccagaa aagcgagaaa gtgatccggc gctacagagt
     2941 gcaggatgcc ctgctgtttc tgctggccaa aaagaccctg accgaactgg ccgatttcga
     3001 cggcgagagg ttcaaactga aagaaatcat gcccgacgcc gagaagggaa tcctgagcga
     3061 gatcatgccc atgagcttca ccttcgagaa aggcggcaag aagtacacca tcaccagcga
     3121 gggcatgaag ctgaagaact acggcgactt ctttgtgctg gctagcgaca agaggatcgg
     3181 caacctgctg gaactcgtgg gcagcgacat cgtgtccaaa gaggatggat cccttcaact
     3241 gcctccactt gaaagactga cactgtccgg aggatctagc ggaggctcct ctggctctga
     3301 gacacctggc acaagcgaga gcgcaacacc tgaaagcagc gggggcagca gcggggggtc
     3361 agtgggtggc gactccagcg cggatcgtct gttcccgccg cagtggatct gctgcgacat
     3421 tcgttacctg gatgtgagca tcctgggcaa gtttgctgtt gtgatggccg acccgccgtg
     3481 ggatattcac atggagctgc cgtatggcac cctgaccgac gatgaaatgc gtcgcctgaa
     3541 catcccggtg ctgcaggacg atggtttcct gtttctgtgg gtgacgggtc gtgctatgga
     3601 gctgggtcgc gaatgcctga acctgtgggg ctacgagcgt gtggacgaaa tcatttgggt
     3661 gaaaaccaac cagctgcagc gcatcattcg taccggtcgt accggtcact ggctgaacca
     3721 cggcaaggag cactgcctgg tgggtgtgaa aggcaacccg cagggtttca accagggcct
     3781 ggactgcgat gtgatcgtgg ctgaagtgcg tagcaccagc cacaagccgg acgagatcta
     3841 tggtatgatt gaacgtctga gcccgggcac ccgcaaaatt gagctgtttg gtcgcccgca
     3901 caacgtgcag ccgaactgga tcaccctggg caaccagctg gacggcattc acctgctgga
     3961 cccggatgtg gtggcccgtt tcaagcagcg ttacccggat ggtatcatta gcaagccgaa
     4021 aaacctgggt ggtagcggcg gcagcggtgg tagcggcggt tccggtggta gcggcggtag
     4081 cggtggcagc ggcggtagcg gtggtagcgg tagcggtcag agcctgaacc cgcacaacga
     4141 ctattgccag cacttcgtgg ataccggtca ccgtccgcag aactttatcc gcgacgtggg
     4201 tctggcggat cgtttcgagg aatacccgaa gctgcgtgag ctgatccgcc tgaaggacga
     4261 actgattgct aaaagcaaca ccccgccgat gtatctgcag gcggacatcg aggctttcga
     4321 tattcgtgaa ctgaccccga aatttgatgt gatcctgctg gagccgccgc tggaggaata
     4381 ctatcgcgaa accggcatca ccgcgaacga aaagtgctgg acctgggacg atattatgaa
     4441 actggagatc gacgaaattg ccgcgccgcg tagcttcatt tttctgtggt gcggtagcgg
     4501 cgagggcctg gacctgggtc gcgtgtgcct gcgtaaatgg ggctaccgtc gttgcgaaga
     4561 tatctgctgg attaagacca acaaaaacaa cccgggcaag accaaaaccc tggatccgaa
     4621 ggccgtgttt cagcgtacca aagagcactg cctgatgggt atcaagggca ccgtgaaacg
     4681 cagcaccgac ggcgatttca tccacgcgaa cgtggacatt gatctgatca ttaccgagga
     4741 accggagatc ggtaacattg agaagccggt ggaaatcttc cacatcattg aacacttttg
     4801 cctgggccgt cgccgtctgc acctgtttgg tcgtgacagc accattcgtc caggttggct
     4861 gaccgtgggt ccgaccctga ccaacagcaa ctacaacgcc gaaacctacg cgagctattt
     4921 cagcgcgccg aacagctatc tgaccggctg caccgaggaa atcgaacgcc tgcgtccgaa
     4981 aagcccacca ccaaagagca aaagcgatcg tggtggtggt gctccacgtg gtggcggtcg
     5041 cggcggtacc agcgccggtc gcggccgtga gcgcaaccgt agcaacttcc gtggtgaacg
     5101 tggtggtttt cgtggcggtc gcggtggtgc tcaccgcggc ggttttccac cacgctaacc
     5161 ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct
     5221 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     5281 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     5341 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     5401 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     5461 ggctcgatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     5521 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5581 aaagcctagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5641 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5701 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5761 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5821 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5881 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5941 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6001 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6061 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6121 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6181 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6241 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6301 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6361 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6421 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6481 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacac tcagtggaac
     6541 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6601 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6661 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6721 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6781 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6841 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6901 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6961 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     7021 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7081 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7141 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7201 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7261 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7321 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7381 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7441 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7501 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7561 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7621 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcga
     7681 tctcccgatc ccctagggtc gactctcagt acaatctgct ctgatgccgc atagttaagc
     7741 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     7801 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     7861 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
     7921 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     7981 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     8041 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     8101 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     8161 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     8221 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
//
