LOCUS Exported 6240 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Empty TALE vector driven by EF1a promoter, no Puro, contains DNMT3a catalytic domain. ACCESSION . VERSION . KEYWORDS LLP245 pEF1a-TALE-DNMT3a-Wt SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6240) AUTHORS Pflueger C, Tan D, Swain T, Nguyen TV, Pflueger J, Nefzger C, Polo JM, Ford E, Lister R TITLE A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs. JOURNAL Genome Res. 2018 Jun 15. pii: gr.233049.117. doi: 10.1101/gr.233049.117. PUBMED 29907613 REFERENCE 2 (bases 1 to 6240) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6240 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 281..1459 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 512..1450 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1407..1427 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 1475..1501 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1505..1531 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1535..1561 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1571..1591 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2303..2323 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2366..3298 /codon_start=1 /product="catalytic domain of DNA methyltransferase 3A" /label=DNMT3A catalytic domain /translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVS DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR LARQRLLGRSWSVPVIRHLFAPLKEYFACV" intron 3452..3681 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 3664..3683 /label=pMT2-F /note="Synthetic intron, forward primer" polyA_signal 3860..3981 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3897..3916) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(4083..4099) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4083..4099) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4096..4118) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4107..4123 /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(4131..4161) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4176..4197 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4314..4331) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4485..5073) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4565..4584) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5244..6104) /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 5867..5886 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6105..6209) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg tgcaaagatg gataaagttt taaacagaga ggaatctttg 241 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg 301 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac 361 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg 421 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct 481 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc 541 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta 601 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc 661 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg 721 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat 781 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc 841 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg 901 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg 961 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat 1021 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg 1081 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg 1141 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg 1201 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag 1261 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg 1321 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct 1381 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct 1441 tccatttcag gtgtcgtgac gtacggccac catgtacccc tatgacgtgc ctgattacgc 1501 cgcttatcct tacgatgtgc ccgactatgc agcctaccca tacgatgtgc ccgactacgc 1561 cgcaatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgtaga 1621 tttgagaact ttgggatatt cacagcagca gcaggaaaag atcaagccca aagtgaggtc 1681 gacagtcgcg cagcatcacg aagccctggt gggtcatggg tttacacatg cccacatcgt 1741 agccttgtcg cagcaccctg cagcccttgg cacggtcgcc gtcaagtacc aggacatgat 1801 tgcggcgttg ccggaagcca cacatgaggc gatcgtcggt gtggggaaac agtggagcgg 1861 agcccgcgcc cttgaggccc tgttgacggt cgcgggagag ctgagagggc ctccccttca 1921 gctggacacg ggccagttgc tgaagatcgc gaagcgggga ggagtcacgg cggtcgaggc 1981 ggtgcacgcg tggcgcaatg cgctcacggg agcacccctc aacctgacag agacgctggc 2041 tgatgggcgt ctcggaggca gacccgcact ggagtcaatt gtggcccagc tttcgaggcc 2101 ggaccccgcg ctggccgcac tcactaatga tcatcttgta gcgctggcct gcctcggcgg 2161 acgacccgcc ttggatgcgg tgaagaaggg gctcccgcac gcgcctgcat tgattaagcg 2221 gaccaacaga aggattcccg agaggacatc acatcgagtg gcaggtggcg gagggtctgg 2281 gggaggcggt agtcaggcca gcccgaaaaa gaaaagaaaa gtgggacggg cgaaaggagg 2341 cggtggctca ggcggtgggg gaagcccctc ccggctccag atgttcttcg ctaataacca 2401 cgaccaggaa tttgaccctc caaaggttta cccacctgtc ccagctgaga agaggaagcc 2461 catccgggtg ctgtctctct ttgatggaat cgctacaggg ctcctggtgc tgaaggactt 2521 gggcattcag gtggaccgct acattgcctc ggaggtgtgt gaggactcca tcacggtggg 2581 catggtgcgg caccagggga agatcatgta cgtcggggac gtccgcagcg tcacacagaa 2641 gcatatccag gagtggggcc cattcgatct ggtgattggg ggcagtccct gcaatgacct 2701 ctccatcgtc aaccctgctc gcaagggcct ctacgagggc actggccggc tgttctttga 2761 gttctaccgc ctcctgcatg atgcgcggcc caaggaggga gatgatcgcc ccttcttctg 2821 gctctttgag aatgtggtgg ccatgggcgt tagtgacaag agggacatct cgcgatttct 2881 cgagtccaac cctgtgatga ttgatgccaa agaagtgtca gctgcacaca gggcccgcta 2941 cttctggggt aaccttcccg gtatgaacag gccgttggca tccactgtga atgataagct 3001 ggagctgcag gagtgtctgg agcatggcag gatagccaag ttcagcaaag tgaggaccat 3061 tactacgagg tcaaactcca taaagcaggg caaagaccag cattttcctg tcttcatgaa 3121 tgagaaagag gacatcttat ggtgcactga aatggaaagg gtatttggtt tcccagtcca 3181 ctatactgac gtgtccaaca tgagccgctt ggcgaggcag agactgctgg gccggtcatg 3241 gagcgtgcca gtcatccgcc acctcttcgc tccgctgaag gagtattttg cgtgtgtgga 3301 ggtgcacacc aaccaagatc cgcttgatgc tgaagttcat acaaaccagg atcccctcga 3361 tgccgaagtc catactaatc aggacccact ggacgcatga gggggaggct aactggaatt 3421 aattcgctgt ctgcgagggc cagctgttgg ggtgagtact ccctctcaaa agcgggcatg 3481 acttctgcgc taagattgtc agtttccaaa aacgaggagg atttgatatt cacctggccc 3541 gcggtgatgc ctttgagggt ggccgcgtcc atctggtcag aaaagacaat ctttttgttg 3601 tcaagcttga ggtgtggcag gcttgagatc tggccataca cttgagtgac aatgacatcc 3661 actttgcctt tctctccaca ggtgtccact cccaggtcca actgcaggtc gatttaaatg 3721 ggaattccac agcggccgca tgtctagata actgatcata atcagccata ccacatttgt 3781 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat 3841 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa 3901 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 3961 caaactcatc aatgtatctt aatgcgtcga gtgcattcta gttgtggttt gtccaaactc 4021 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca 4081 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 4141 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 4201 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 4261 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 4321 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 4381 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 4441 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 4501 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 4561 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 4621 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 4681 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 4741 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 4801 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 4861 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 4921 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 4981 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 5041 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 5101 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5161 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 5221 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 5281 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 5341 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 5401 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 5461 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 5521 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 5581 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 5641 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 5701 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 5761 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 5821 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 5881 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 5941 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 6001 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 6061 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 6121 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 6181 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc //