LOCUS       sequence-307628-        8280 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 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-inactive M3M14nes
FEATURES             Location/Qualifiers
     source          1..8280
                     /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             3603..3629
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             5528..5545
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5568..5585)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5574..5798
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5869..5885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5869..5885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5882..5904)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5893..5909
                     /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(5917..5923,5924..5941,5942..5947))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5962..5983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6100..6117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6271..6859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6351..6370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7030..7821,7822..7890))
                     /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     7653..7672
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7891..7995)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8074..8093)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8265..8280,1..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 catgaacatc cccgctctgg
      661 tggaaaacca gaagaagtac tttggcacct acagcgtgat ggccatgctg aacgctcaga
      721 ccgtgctgga ccacatccag aaggtggccg atattgaggg cgagcagaac gagaacaacg
      781 agaatctgtg gtttcacccc gtgatgagcc acctgtacaa cgccaagaac ggctacgaca
      841 agcagcccga gaaaaccatg ttcatcatcg agcggctgca gagctacttc ccattcctga
      901 agatcatggc cgagaaccag agagagtaca gcaacggcaa gtacaagcag aaccgcgtgg
      961 aagtgaacag caacgacatc ttcgaggtgc tgaagcgcgc cttcggcgtg ctgaagatgt
     1021 acagggacct gaccaacgca tacaagacct acgaggaaaa gctgaacgac ggctgcgagt
     1081 tcctgaccag cacagagcaa cctctgagcg gcatgatcaa caactactac acagtggccc
     1141 tgcggaacat gaacgagaga tacggctaca agacagagga cctggccttc atccaggaca
     1201 agcggttcaa gttcgtgaag gacgcctacg gcaagaaaaa gtcccaagtg aataccggat
     1261 tcttcctgag cctgcaggac tacaacggcg acacacagaa gaagctgcac ctgagcggag
     1321 tgggaatcgc cctgctgatc tgcctgttcc tggacaagca gtacatcaac atctttctga
     1381 gcaggctgcc catcttctcc agctacaatg cccagagcga ggaacggcgg atcatcatca
     1441 gatccttcgg catcaacagc atcaagctgc ccaaggaccg gatccacagc gagaagtcca
     1501 acaagagcgt ggccatggat atgctcaacg aagtgaagcg gtgccccgac gagctgttca
     1561 caacactgtc tgccgagaag cagtcccggt tcagaatcat cagcgacgac cacaatgaag
     1621 tgctgatgaa gcggagcagc gacagattcg tgcctctgct gctgcagtat atcgattacg
     1681 gcaagctgtt cgaccacatc aggttccacg tgaacatggg caagctgaga tacctgctga
     1741 aggccgacaa gacctgcatc gacggccaga ccagagtcag agtgatcgag cagcccctga
     1801 acggcttcgg cagactggaa gaggccgaga caatgcggaa gcaagagaac ggcaccttcg
     1861 gcaacagcgg catccggatc agagacttcg agaacatgaa gcgggacgac gccaatcctg
     1921 ccaactatcc ctacatcgtg gacacctaca cacactacat cctggaaaac aacaaggtcg
     1981 agatgtttat caacgacaaa gaggacagcg ccccactgct gcccgtgatc gaggatgata
     2041 gatacgtggt caagacaatc cccagctgcc ggatgagcac cctggaaatt ccagccatgg
     2101 ccttccacat gtttctgttc ggcagcaaga aaaccgagaa gctgatcgtg gacgtgcaca
     2161 accggtacaa gagactgttc caggccatgc agaaagaaga agtgaccgcc gagaatatcg
     2221 ccagcttcgg aatcgccgag agcgacctgc ctcagaagat cctggatctg atcagcggca
     2281 atgcccacgg caaggatgtg gacgccttca tcagactgac cgtggacgac atgctgaccg
     2341 acaccgagcg gagaatcaag agattcaagg acgaccggaa gtccattcgg agcgccgaca
     2401 acaagatggg aaagagaggc ttcaagcaga tctccacagg caagctggcc gacttcctgg
     2461 ccaaggacat cgtgctgttt cagcccagcg tgaacgatgg cgagaacaag atcaccggcc
     2521 tgaactaccg gatcatgcag agcgccattg ccgtgtacga tagcggcgac gattacgagg
     2581 ccaagcagca gttcaagctg atgttcgaga aggcccggct gatcggcaag ggcacaacag
     2641 agcctcatcc atttctgtac aaggtgttcg cccgcagcat ccccgccaat gccgtcgagt
     2701 tctacgagcg ctacctgatc gagcggaagt tctacctgac cggcctgtcc aacgagatca
     2761 agaaaggcaa cagagtggat gtgcccttca tccggcggga ccagaacaag tggaaaacac
     2821 ccgccatgaa gaccctgggc agaatctaca gcgaggatct gcccgtggaa ctgcccagac
     2881 agatgttcga caatgagatc aagtcccacc tgaagtccct gccacagatg gaaggcatcg
     2941 acttcaacaa tgccaacgtg acctatctga tcgccgagta catgaagaga gtgctggacg
     3001 acgacttcca gaccttctac cagtggaacc gcaactaccg gtacatggac atgcttaagg
     3061 gcgagtacga cagaaagggc tccctgcagc actgcttcac cagcgtggaa gagagagaag
     3121 gcctctggaa agagcgggcc tccagaacag agcggtacag aaagcaggcc agcaacaaga
     3181 tccgcagcaa ccggcagatg agaaacgcca gcagcgaaga gatcgagaca atcctggata
     3241 agcggctgag caacagccgg aacgagtacc agaaaagcga gaaagtgatc cggcgctaca
     3301 gagtgcagga tgccctgctg tttctgctgg ccaaaaagac cctgaccgaa ctggccgatt
     3361 tcgacggcga gaggttcaaa ctgaaagaaa tcatgcccga cgccgagaag ggaatcctga
     3421 gcgagatcat gcccatgagc ttcaccttcg agaaaggcgg caagaagtac accatcacca
     3481 gcgagggcat gaagctgaag aactacggcg acttctttgt gctggctagc gacaagagga
     3541 tcggcaacct gctggaactc gtgggcagcg acatcgtgtc caaagaggat ggatcccttc
     3601 aactgcctcc acttgaaaga ctgacactgt ccggaggatc tagcggaggc tcctctggct
     3661 ctgagacacc tggcacaagc gagagcgcaa cacctgaaag cagcgggggc agcagcgggg
     3721 ggtcagtggg tggcgactcc agcgcggatc gtctgttccc gccgcagtgg atctgctgcg
     3781 acattcgtta cctggatgtg agcatcctgg gcaagtttgc tgttgtgatg gccgccccgc
     3841 cgtgggatat tcacatggag ctgccgtatg gcaccctgac cgacgatgaa atgcgtcgcc
     3901 tgaacatccc ggtgctgcag gacgatggtt tcctgtttct gtgggtgacg ggtcgtgcta
     3961 tggagctggg tcgcgaatgc ctgaacctgt ggggctacga gcgtgtggac gaaatcattt
     4021 gggtgaaaac caaccagctg cagcgcatca ttcgtaccgg tcgtaccggt cactggctga
     4081 accacggcaa ggagcactgc ctggtgggtg tgaaaggcaa cccgcagggt ttcaaccagg
     4141 gcctggactg cgatgtgatc gtggctgaag tgcgtagcac cagccacaag ccggacgaga
     4201 tctatggtat gattgaacgt ctgagcccgg gcacccgcaa aattgagctg tttggtcgcc
     4261 cgcacaacgt gcagccgaac tggatcaccc tgggcaacca gctggacggc attcacctgc
     4321 tggacccgga tgtggtggcc cgtttcaagc agcgttaccc ggatggtatc attagcaagc
     4381 cgaaaaacct gggtggtagc ggcggcagcg gtggtagcgg cggttccggt ggtagcggcg
     4441 gtagcggtgg cagcggcggt agcggtggta gcggtagcgg tcagagcctg aacccgcaca
     4501 acgactattg ccagcacttc gtggataccg gtcaccgtcc gcagaacttt atccgcgacg
     4561 tgggtctggc ggatcgtttc gaggaatacc cgaagctgcg tgagctgatc cgcctgaagg
     4621 acgaactgat tgctaaaagc aacaccccgc cgatgtatct gcaggcggac atcgaggctt
     4681 tcgatattcg tgaactgacc ccgaaatttg atgtgatcct gctggagccg ccgctggagg
     4741 aatactatcg cgaaaccggc atcaccgcga acgaaaagtg ctggacctgg gacgatatta
     4801 tgaaactgga gatcgacgaa attgccgcgc cgcgtagctt catttttctg tggtgcggta
     4861 gcggcgaggg cctggacctg ggtcgcgtgt gcctgcgtaa atggggctac cgtcgttgcg
     4921 aagatatctg ctggattaag accaacaaaa acaacccggg caagaccaaa accctggatc
     4981 cgaaggccgt gtttcagcgt accaaagagc actgcctgat gggtatcaag ggcaccgtga
     5041 aacgcagcac cgacggcgat ttcatccacg cgaacgtgga cattgatctg atcattaccg
     5101 aggaaccgga gatcggtaac attgagaagc cggtggaaat cttccacatc attgaacact
     5161 tttgcctggg ccgtcgccgt ctgcacctgt ttggtcgtga cagcaccatt cgtccaggtt
     5221 ggctgaccgt gggtccgacc ctgaccaaca gcaactacaa cgccgaaacc tacgcgagct
     5281 atttcagcgc gccgaacagc tatctgaccg gctgcaccga ggaaatcgaa cgcctgcgtc
     5341 cgaaaagccc accaccaaag agcaaaagcg atcgtggtgg tggtgctcca cgtggtggcg
     5401 gtcgcggcgg taccagcgcc ggtcgcggcc gtgagcgcaa ccgtagcaac ttccgtggtg
     5461 aacgtggtgg ttttcgtggc ggtcgcggtg gtgctcaccg cggcggtttt ccaccacgct
     5521 aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
     5581 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5641 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     5701 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     5761 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     5821 ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5881 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5941 gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6001 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6061 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6121 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6181 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6721 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6841 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acactcagtg
     6901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6961 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7021 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7081 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7141 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7201 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7261 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7321 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7381 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7441 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7501 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7561 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7621 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7681 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7741 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7801 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7861 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7921 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7981 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     8041 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     8101 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     8161 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     8221 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
//
