LOCUS pCMV-dCas13-M3nls 7434 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Targeted m6A RNA methylation in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pCMV-dCas13-M3nls
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 May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7434
/organism="synthetic DNA construct"
/mol_type="other DNA"
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(4707..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(5820..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
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