LOCUS Exported 10381 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Targeted DNA methylation. ACCESSION . VERSION . KEYWORDS pSt1374m-EF1alpha-N-NLS-DNMT3L-L-DNMT3Amut-L-dcas9-NLS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10381) AUTHORS Lu Z, Liu Z, Mao W, Wang X, Zheng X, Chen S, Cao B, Huang S, Zhang X, Zhou T, Zhang Y, Huang X, Sun Q, Li JD TITLE Locus-specific DNA methylation of Mecp2 promoter leads to autism-like phenotypes in mice. JOURNAL Cell Death Dis. 2020 Feb 3;11(2):85. doi: 10.1038/s41419-020-2290-x. PUBMED 32015323 REFERENCE 2 (bases 1 to 10381) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10381 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 11..1191 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 241..1183 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1140..1160 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" primer_bind 1241..1260 /label=T7 /note="T7 promoter, forward primer" promoter 1241..1259 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1288..1308 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1963..1980 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 6997..7017 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7048..7104 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 7105..7815 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(7250..7268) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 7518..7537 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(7835..7852) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 7841..8065 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(8227..8244) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(8398..8986) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(8478..8497) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(9157..10017) /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 9780..9799 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10018..10122) /gene="bla" /label=AmpR promoter primer_bind complement(10197..10216) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 cgcgtgtgag gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag 61 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac 121 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat 181 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag 241 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg 301 ccttgaatta cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt 361 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg 421 agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc 481 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac 541 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc 601 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag 661 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc 721 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc 781 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag 841 ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa 901 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc 961 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg 1021 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg 1081 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct 1141 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt ggagctctct 1201 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg 1261 agacccaagc tggctagcac catgggacct aagaaaaaga ggaaggtggc ggccgctggc 1321 ggcagcatgt tcgaaaccgt gcctgtgtgg aggagacagc cagtccgggt gctgtccctt 1381 tttgaagaca tcaagaaaga gctgacgagt ttgggctttt tggaaagtgg ttctgacccg 1441 ggacaactga agcatgtggt tgatgtcaca gacacagtga ggaaggatgt ggaggagtgg 1501 ggacccttcg atcttgtgta cggcgccaca cctcccctgg gccacacctg tgaccgtcct 1561 cccagctggt acctgttcca gttccaccgg ctcctgcagt acgcacggcc caagccaggc 1621 agccccaggc ccttcttctg gatgttcgtg gacaatctgg tgctgaacaa ggaagacctg 1681 gacgtcgcat ctcgcttcct ggagatggag ccagtcacca tcccagatgt ccacggcgga 1741 tccttgcaga atgctgtccg cgtgtggagc aacatcccag ccataaggag cagcaggcac 1801 tgggctctgg tttcggaaga agaattgtcc ctgctggccc agaacaagca gagctcgaag 1861 ctcgcggcca agtggcccac caagctggtg aagaactgct ttctccccct aagagaatat 1921 ttcaagtatt tttcaagcag cgggcgcagc ttcagcagcg gactagtgcc ccgcggcagc 1981 cacccagctg agaagaggaa gcccatccgg gtgctgtctc tctttgatgg aatcgctaca 2041 gggctcctgg tgctgaagga cttgggcatt caggtggacc gctacattgc ctcggaggtg 2101 tgtgaggact ccatcacggt gggcatggtg cggcaccagg ggaagatcat gtacgtcggg 2161 gacgtccgca gcgtcacaca gaagcatatc caggagtggg gcccattcga tctggtgatt 2221 gggggcagtc cctccaatga cctctccatc gtcaaccctg ctcgcaaggg cctctacgag 2281 ggcactggcc ggctcttctt tgagttctac cgcctcctgc atgatgcgcg gcccaaggag 2341 ggagatgatc gccccttctt ctggctcttt gagaatgtgg tggccatggg cgttagtgac 2401 aagagggaca tctcgcgatt tctcgagtcc aaccctgtga tgattgatgc caaagaagtg 2461 tcagctgcac acagggcccg ctacttctgg ggtaaccttc ccggtatgaa caggccgttg 2521 gcatccactg tgaatgataa gctggagctg caggagtgtc tggagcatgg caggatagcc 2581 aagttcagca aagtgaggac cattactacg aggtcaaact ccataaagca gggcaaagac 2641 cagcattttc ctgtcttcat gaatgagaaa gaggacatct tatggtgcac tgaaatggaa 2701 agggtatttg gtttcccagt ccactatact gacgtctcca acatgagccg cttggcgagg 2761 cagagactgc tgggccggtc atggagcgtg ccagtcatcc gccacctctt cgctccgctg 2821 aaggagtatt ttgcgtgtgt gagcggcagc gagacccccg gtaccagcga gagcgccacc 2881 cccgagagcg acaagaaata ctctattgga ctggctatcg ggacaaactc cgttggctgg 2941 gccgtcataa ccgacgagta taaggtgcca agcaagaaat tcaaggtgct gggtaatact 3001 gaccgccatt caatcaagaa gaacctgatc ggagcactcc tcttcgactc cggtgaaacc 3061 gctgaagcta ctcggctgaa gcggaccgca aggcggagat acacccgccg caagaatcgg 3121 atatgttatc tgcaagagat ctttagcaac gaaatggcta aggtggacga ctccttcttt 3181 caccgcctgg aagagagctt tctggtggag gaggataaga aacacgagag gcaccctata 3241 ttcggaaata tcgtggatga ggtggcttac catgaaaagt atcctacaat ctaccatctg 3301 aggaagaagc tggtggacag caccgataaa gcagacctga ggctcatcta tctggccctg 3361 gctcatatga taaagtttag aggacacttt ctgatcgagg gcgacctgaa tcccgataat 3421 tccgatgtgg ataaactctt cattcaactg gtgcagacat ataaccaact gttcgaggag 3481 aatcccataa acgcttctgg tgtggatgcc aaggctattc tgtccgctcg gctgtccaag 3541 tcacgcagac tggagaatct gattgcccaa ctgccaggag aaaagaagaa cggcctgttt 3601 gggaacctca tcgccctgag cctgggcctg acacctaact tcaagtccaa ttttgatctg 3661 gccgaagatg ctaaactcca gctctccaag gacacctatg acgatgatct ggacaacctg 3721 ctcgcacaga taggcgacca gtacgccgat ctctttctgg ctgctaagaa tctctccgac 3781 gccattctgc tgagcgacat actccgggtc aacactgaga tcaccaaagc acctctgagc 3841 gcctccatga taaaacgcta tgatgaacac catcaagacc tgactctgct caaagccctc 3901 gtgaggcaac agctgccaga gaagtacaaa gagatattct tcgaccagag caagaatgga 3961 tatgccggat acatcgatgg cggagcatca caggaagaat tttacaagtt catcaaacca 4021 atcctcgaga agatggacgg tactgaagag ctgctggtga agctgaacag ggaggacctg 4081 ctgaggaagc agaggacctt tgataatggc tccattccac atcagataca cctgggagag 4141 ctgcatgcaa tcctccgcag gcaggaggat ttctatcctt tcctgaagga taaccgggag 4201 aagatagaga agatcctgac cttcaggatc ccttattacg tcggccctct ggctagaggc 4261 aactcccgct tcgcttggat gaccaggaaa tctgaggaga caattactcc ttggaacttc 4321 gaagaggtcg tggataaggg cgcaagcgcc cagtcattca tcgaacggat gaccaatttc 4381 gataagaacc tgccaaacga gaaggtcctg cccaaacatt cactcctgta cgagtatttc 4441 accgtctata acgagctgac taaagtgaag tacgtgaccg agggcatgag gaagcctgcc 4501 ttcctgtccg gagagcagaa gaaggctatc gttgatctgc tcttcaagac taatagaaag 4561 gtgacagtga agcagctcaa ggaggattac tttaagaaga tcgaatgctt tgactcagtg 4621 gaaatctctg gcgtggagga ccgctttaat gccagcctgg gcacttacca tgatctgctg 4681 aagataatca aagacaaaga tttcctcgat aatgaggaga acgaggacat cctggaagat 4741 atcgtgctga ccctgactct gttcgaggat agagagatga tcgaagagcg cctgaagacc 4801 tatgcccatc tgtttgacga taaagtcatg aaacagctca agcggcggcg ctacactggg 4861 tggggtagac tctccaggaa actcataaac ggcatccgcg acaaacagag cggaaagacc 4921 atcctggatt tcctgaaatc cgacggattc gctaacagga acttcatgca actgattcac 4981 gatgactctc tgacatttaa agaggacatc cagaaggcac aggtgagcgg tcaaggcgac 5041 agcctgcacg agcacatcgc caacctcgct ggatcacccg ccataaagaa gggaatactg 5101 cagacagtca aggtcgtgga cgaactcgtc aaagtgatgg gtcggcacaa gccagagaat 5161 atcgttatcg aaatggcaag ggagaaccaa accacccaga agggccagaa gaactctcgg 5221 gaacggatga aaagaatcga agagggaatt aaggagctgg gatctcagat actgaaggag 5281 caccctgtgg agaatacaca gctccagaac gagaaactct acctgtacta cctccagaac 5341 gggcgggaca tgtacgttga ccaggaactc gacatcaacc ggctgtccga ttatgacgtg 5401 gacgctattg ttccacagtc cttcctcaaa gatgactcca ttgacaacaa ggtgctgacc 5461 agatccgata aggcccgcgg taagtctgac aatgttccat cagaagaggt ggtcaagaag 5521 atgaagaatt actggcggca gctcctcaac gccaaactga tcacccagcg gaagtttgac 5581 aatctgacta aggcagaaag aggaggtctg agcgaactcg acaaggccgg ctttattaag 5641 aggcaactgg tcgaaacacg ccagattacc aaacacgtgg cacaaatcct cgactctagg 5701 atgaacacta agtacgatga gaacgataag ctgatcaggg aagtgaaagt gataactctg 5761 aagagcaagc tggtgtctga cttccggaag gactttcaat tctacaaagt tcgcgaaata 5821 aacaattacc atcatgctca cgatgcctat ctcaatgctg tcgttggcac cgccctgatc 5881 aagaaatacc ctaaactgga gtctgagttc gtgtacggtg actataaagt ctacgatgtg 5941 aggaagatga tagcaaagtc tgagcaagag attggcaaag ccaccgccaa gtacttcttc 6001 tactctaata tcatgaattt ctttaagact gagataaccc tggctaacgg cgaaatccgg 6061 aagcgcccac tgatcgaaac aaacggagaa acaggagaaa tcgtgtggga taaaggcagg 6121 gacttcgcaa ctgtgcggaa ggtgctgtcc atgccacaag tcaatatcgt gaagaagacc 6181 gaagtgcaga ccggcggatt ctcaaaggag agcatcctgc caaagcggaa ctctgacaag 6241 ctgatcgcca ggaagaaaga ttgggaccca aagaagtatg gcggtttcga ttcccctaca 6301 gtggcttatt ccgttctggt cgtggcaaaa gtggagaaag gcaagtccaa gaaactcaag 6361 tctgttaagg agctgctcgg aattactatt atggagagat ccagcttcga gaagaatcca 6421 atcgatttcc tggaagctaa gggctataaa gaagtgaaga aagatctcat catcaaactg 6481 cccaagtact ctctctttga gctggagaat ggtaggaagc ggatgctggc ctccgccgga 6541 gagctgcaga aaggaaacga gctggctctg ccctccaaat acgtgaactt cctgtatctg 6601 gcctcccact acgagaaact caaaggtagc cctgaagaca atgagcagaa gcaactcttt 6661 gttgagcaac ataaacacta cctggacgaa atcattgaac agattagcga gttcagcaag 6721 cgggttattc tggccgatgc aaacctcgat aaagtgctga gcgcatataa taagcacagg 6781 gacaagccaa ttcgcgaaca agcagagaat attatccacc tctttactct gactaatctg 6841 ggcgctcctg ctgccttcaa gtatttcgat acaactattg acaggaagcg gtacacctct 6901 accaaagaag ttctcgatgc caccctgata caccagtcaa ttaccggact gtacgagact 6961 cgcatcgacc tgtctcagct cggcggcgac ggttctccca agaagaagag gaaagtctcg 7021 agcggtggag ctgcaggagg atccggcgcc acgaacttct ctctgttaaa gcaagcagga 7081 gacgtggaag aaaaccccgg tcctatggtg agcaagggcg aggaggataa catggccatc 7141 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc 7201 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag 7261 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac 7321 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc 7381 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg 7441 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc 7501 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc 7561 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg 7621 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg 7681 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac 7741 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac 7801 gagctgtaca agtaagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag 7861 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 7921 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 7981 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 8041 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg 8101 gggtatcccc attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 8161 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 8221 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 8281 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 8341 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 8401 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 8461 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 8521 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 8581 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 8641 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 8701 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 8761 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 8821 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 8881 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 8941 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 9001 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 9061 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 9121 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 9181 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 9241 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 9301 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 9361 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 9421 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 9481 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 9541 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 9601 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 9661 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 9721 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 9781 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 9841 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 9901 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 9961 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 10021 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 10081 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 10141 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca 10201 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc 10261 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc 10321 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat 10381 a //