LOCUS Exported 10381 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Targeted DNA methylation. ACCESSION . VERSION . KEYWORDS pSt1374m-EF1alpha-N-NLS-DNMT3L-L-DNMT3A-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 1..1181 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 231..1173 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1130..1150 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" primer_bind 1231..1250 /label=T7 /note="T7 promoter, forward primer" promoter 1231..1249 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1278..1298 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1953..1970 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 6987..7007 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7038..7094 /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 7095..7805 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTRGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind 7508..7527 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(7825..7842) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 7831..8055 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(8217..8234) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(8388..8976) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(8468..8487) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(9147..10007) /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 9770..9789 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10008..10112) /gene="bla" /label=AmpR promoter primer_bind complement(10187..10206) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 61 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 121 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 181 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 241 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 301 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg 361 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc 421 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct 481 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc 541 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg 601 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg 661 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg 721 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca 781 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg 841 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt 901 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc 961 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg 1021 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg 1081 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag 1141 acagtggttc aaagtttttt tcttccattt caggtgtcgt ggagctctct ggctaactag 1201 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc 1261 tggctagcac catgggacct aagaaaaaga ggaaggtggc ggccgctggc ggcagcatgt 1321 tcgaaaccgt gcctgtgtgg aggagacagc cagtccgggt gctgtccctt tttgaagaca 1381 tcaagaaaga gctgacgagt ttgggctttt tggaaagtgg ttctgacccg ggacaactga 1441 agcatgtggt tgatgtcaca gacacagtga ggaaggatgt ggaggagtgg ggacccttcg 1501 atcttgtgta cggcgccaca cctcccctgg gccacacctg tgaccgtcct cccagctggt 1561 acctgttcca gttccaccgg ctcctgcagt acgcacggcc caagccaggc agccccaggc 1621 ccttcttctg gatgttcgtg gacaatctgg tgctgaacaa ggaagacctg gacgtcgcat 1681 ctcgcttcct ggagatggag ccagtcacca tcccagatgt ccacggcgga tccttgcaga 1741 atgctgtccg cgtgtggagc aacatcccag ccataaggag cagcaggcac tgggctctgg 1801 tttcggaaga agaattgtcc ctgctggccc agaacaagca gagctcgaag ctcgcggcca 1861 agtggcccac caagctggtg aagaactgct ttctccccct aagagaatat ttcaagtatt 1921 tttcaagcag cgggcgcagc ttcagcagcg gactagtgcc ccgcggcagc cacccagctg 1981 agaagaggaa gcccatccgg gtgctgtctc tctttgatgg aatcgctaca gggctcctgg 2041 tgctgaagga cttgggcatt caggtggacc gctacattgc ctcggaggtg tgtgaggact 2101 ccatcacggt gggcatggtg cggcaccagg ggaagatcat gtacgtcggg gacgtccgca 2161 gcgtcacaca gaagcatatc caggagtggg gcccattcga tctggtgatt gggggcagtc 2221 cctgcaatga cctctccatc gtcaaccctg ctcgcaaggg cctctacgag ggcactggcc 2281 ggctcttctt tgagttctac cgcctcctgc atgatgcgcg gcccaaggag ggagatgatc 2341 gccccttctt ctggctcttt gagaatgtgg tggccatggg cgttagtgac aagagggaca 2401 tctcgcgatt tctcgagtcc aaccctgtga tgattgatgc caaagaagtg tcagctgcac 2461 acagggcccg ctacttctgg ggtaaccttc ccggtatgaa caggccgttg gcatccactg 2521 tgaatgataa gctggagctg caggagtgtc tggagcatgg caggatagcc aagttcagca 2581 aagtgaggac cattactacg aggtcaaact ccataaagca gggcaaagac cagcattttc 2641 ctgtcttcat gaatgagaaa gaggacatct tatggtgcac tgaaatggaa agggtatttg 2701 gtttcccagt ccactatact gacgtctcca acatgagccg cttggcgagg cagagactgc 2761 tgggccggtc atggagcgtg ccagtcatcc gccacctctt cgctccgctg aaggagtatt 2821 ttgcgtgtgt gagcggcagc gagacccccg gtaccagcga gagcgccacc cccgagagcg 2881 acaagaaata ctctattgga ctggctatcg ggacaaactc cgttggctgg gccgtcataa 2941 ccgacgagta taaggtgcca agcaagaaat tcaaggtgct gggtaatact gaccgccatt 3001 caatcaagaa gaacctgatc ggagcactcc tcttcgactc cggtgaaacc gctgaagcta 3061 ctcggctgaa gcggaccgca aggcggagat acacccgccg caagaatcgg atatgttatc 3121 tgcaagagat ctttagcaac gaaatggcta aggtggacga ctccttcttt caccgcctgg 3181 aagagagctt tctggtggag gaggataaga aacacgagag gcaccctata ttcggaaata 3241 tcgtggatga ggtggcttac catgaaaagt atcctacaat ctaccatctg aggaagaagc 3301 tggtggacag caccgataaa gcagacctga ggctcatcta tctggccctg gctcatatga 3361 taaagtttag aggacacttt ctgatcgagg gcgacctgaa tcccgataat tccgatgtgg 3421 ataaactctt cattcaactg gtgcagacat ataaccaact gttcgaggag aatcccataa 3481 acgcttctgg tgtggatgcc aaggctattc tgtccgctcg gctgtccaag tcacgcagac 3541 tggagaatct gattgcccaa ctgccaggag aaaagaagaa cggcctgttt gggaacctca 3601 tcgccctgag cctgggcctg acacctaact tcaagtccaa ttttgatctg gccgaagatg 3661 ctaaactcca gctctccaag gacacctatg acgatgatct ggacaacctg ctcgcacaga 3721 taggcgacca gtacgccgat ctctttctgg ctgctaagaa tctctccgac gccattctgc 3781 tgagcgacat actccgggtc aacactgaga tcaccaaagc acctctgagc gcctccatga 3841 taaaacgcta tgatgaacac catcaagacc tgactctgct caaagccctc gtgaggcaac 3901 agctgccaga gaagtacaaa gagatattct tcgaccagag caagaatgga tatgccggat 3961 acatcgatgg cggagcatca caggaagaat tttacaagtt catcaaacca atcctcgaga 4021 agatggacgg tactgaagag ctgctggtga agctgaacag ggaggacctg ctgaggaagc 4081 agaggacctt tgataatggc tccattccac atcagataca cctgggagag ctgcatgcaa 4141 tcctccgcag gcaggaggat ttctatcctt tcctgaagga taaccgggag aagatagaga 4201 agatcctgac cttcaggatc ccttattacg tcggccctct ggctagaggc aactcccgct 4261 tcgcttggat gaccaggaaa tctgaggaga caattactcc ttggaacttc gaagaggtcg 4321 tggataaggg cgcaagcgcc cagtcattca tcgaacggat gaccaatttc gataagaacc 4381 tgccaaacga gaaggtcctg cccaaacatt cactcctgta cgagtatttc accgtctata 4441 acgagctgac taaagtgaag tacgtgaccg agggcatgag gaagcctgcc ttcctgtccg 4501 gagagcagaa gaaggctatc gttgatctgc tcttcaagac taatagaaag gtgacagtga 4561 agcagctcaa ggaggattac tttaagaaga tcgaatgctt tgactcagtg gaaatctctg 4621 gcgtggagga ccgctttaat gccagcctgg gcacttacca tgatctgctg aagataatca 4681 aagacaaaga tttcctcgat aatgaggaga acgaggacat cctggaagat atcgtgctga 4741 ccctgactct gttcgaggat agagagatga tcgaagagcg cctgaagacc tatgcccatc 4801 tgtttgacga taaagtcatg aaacagctca agcggcggcg ctacactggg tggggtagac 4861 tctccaggaa actcataaac ggcatccgcg acaaacagag cggaaagacc atcctggatt 4921 tcctgaaatc cgacggattc gctaacagga acttcatgca actgattcac gatgactctc 4981 tgacatttaa agaggacatc cagaaggcac aggtgagcgg tcaaggcgac agcctgcacg 5041 agcacatcgc caacctcgct ggatcacccg ccataaagaa gggaatactg cagacagtca 5101 aggtcgtgga cgaactcgtc aaagtgatgg gtcggcacaa gccagagaat atcgttatcg 5161 aaatggcaag ggagaaccaa accacccaga agggccagaa gaactctcgg gaacggatga 5221 aaagaatcga agagggaatt aaggagctgg gatctcagat actgaaggag caccctgtgg 5281 agaatacaca gctccagaac gagaaactct acctgtacta cctccagaac gggcgggaca 5341 tgtacgttga ccaggaactc gacatcaacc ggctgtccga ttatgacgtg gacgctattg 5401 ttccacagtc cttcctcaaa gatgactcca ttgacaacaa ggtgctgacc agatccgata 5461 aggcccgcgg taagtctgac aatgttccat cagaagaggt ggtcaagaag atgaagaatt 5521 actggcggca gctcctcaac gccaaactga tcacccagcg gaagtttgac aatctgacta 5581 aggcagaaag aggaggtctg agcgaactcg acaaggccgg ctttattaag aggcaactgg 5641 tcgaaacacg ccagattacc aaacacgtgg cacaaatcct cgactctagg atgaacacta 5701 agtacgatga gaacgataag ctgatcaggg aagtgaaagt gataactctg aagagcaagc 5761 tggtgtctga cttccggaag gactttcaat tctacaaagt tcgcgaaata aacaattacc 5821 atcatgctca cgatgcctat ctcaatgctg tcgttggcac cgccctgatc aagaaatacc 5881 ctaaactgga gtctgagttc gtgtacggtg actataaagt ctacgatgtg aggaagatga 5941 tagcaaagtc tgagcaagag attggcaaag ccaccgccaa gtacttcttc tactctaata 6001 tcatgaattt ctttaagact gagataaccc tggctaacgg cgaaatccgg aagcgcccac 6061 tgatcgaaac aaacggagaa acaggagaaa tcgtgtggga taaaggcagg gacttcgcaa 6121 ctgtgcggaa ggtgctgtcc atgccacaag tcaatatcgt gaagaagacc gaagtgcaga 6181 ccggcggatt ctcaaaggag agcatcctgc caaagcggaa ctctgacaag ctgatcgcca 6241 ggaagaaaga ttgggaccca aagaagtatg gcggtttcga ttcccctaca gtggcttatt 6301 ccgttctggt cgtggcaaaa gtggagaaag gcaagtccaa gaaactcaag tctgttaagg 6361 agctgctcgg aattactatt atggagagat ccagcttcga gaagaatcca atcgatttcc 6421 tggaagctaa gggctataaa gaagtgaaga aagatctcat catcaaactg cccaagtact 6481 ctctctttga gctggagaat ggtaggaagc ggatgctggc ctccgccgga gagctgcaga 6541 aaggaaacga gctggctctg ccctccaaat acgtgaactt cctgtatctg gcctcccact 6601 acgagaaact caaaggtagc cctgaagaca atgagcagaa gcaactcttt gttgagcaac 6661 ataaacacta cctggacgaa atcattgaac agattagcga gttcagcaag cgggttattc 6721 tggccgatgc aaacctcgat aaagtgctga gcgcatataa taagcacagg gacaagccaa 6781 ttcgcgaaca agcagagaat attatccacc tctttactct gactaatctg ggcgctcctg 6841 ctgccttcaa gtatttcgat acaactattg acaggaagcg gtacacctct accaaagaag 6901 ttctcgatgc caccctgata caccagtcaa ttaccggact gtacgagact cgcatcgacc 6961 tgtctcagct cggcggcgac ggttctccca agaagaagag gaaagtctcg agcggtggag 7021 ctgcaggagg atccggcgcc acgaacttct ctctgttaaa gcaagcagga gacgtggaag 7081 aaaaccccgg tcctatggtg agcaagggcg aggaggataa catggccatc atcaaggagt 7141 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg 7201 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaggg 7261 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg 7321 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct 7381 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact 7441 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct 7501 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt 7561 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg 7621 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg 7681 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg 7741 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca 7801 agtaagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 7861 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 7921 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 7981 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 8041 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc 8101 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 8161 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 8221 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 8281 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 8341 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 8401 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 8461 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 8521 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 8581 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 8641 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 8701 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 8761 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 8821 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 8881 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 8941 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 9001 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 9061 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 9121 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 9181 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 9241 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 9301 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 9361 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 9421 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 9481 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 9541 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 9601 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 9661 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 9721 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 9781 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 9841 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 9901 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 9961 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 10021 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 10081 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 10141 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 10201 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 10261 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 10321 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtgtga 10381 g //