LOCUS Exported 8118 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION TALEN designed to target the human AAVS1 locus . ACCESSION . VERSION . KEYWORDS hAAVS1 1L TALEN SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8118) AUTHORS Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F TITLE A transcription activator-like effector toolbox for genome engineering. JOURNAL Nat Protoc. 2012 Jan 5;7(1):171-92. doi: 10.1038/nprot.2011.431. PUBMED 22222791 REFERENCE 2 (bases 1 to 8118) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8118 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 86..674 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 575..594 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 828..845 /label=L4440 /note="L4440 vector, forward primer" primer_bind 943..960 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 944..960 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 1416..1432 /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)." enhancer 1459..1838 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1839..2042 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1993..2013 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 2087..2106 /label=T7 /note="T7 promoter, forward primer" promoter 2087..2105 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 2107..2116 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2116..2181 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 2188..2208 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4528..5115 /codon_start=1 /product="nonspecific DNA cleavage domain of the FokI endonuclease (Li et al., 1992)" /label=FokI cleavage domain /note="must dimerize to be catalytically active" /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF" primer_bind complement(5133..5150) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5139..5250 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5368..5388) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5373..5702 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5553..5688 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5615..5634 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5751..6776 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" polyA_signal 6906..7027 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6943..6962) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6997..7016 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(7080..7940) /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 7703..7722 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7941..8032) /gene="bla" /label=AmpR promoter primer_bind 8074..8093 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 8099..8118 /label=pENTR-R /note="pENTR vectors, reverse primer" ORIGIN 1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc 61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc 301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc 901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt 961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg 1021 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag 1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg 1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata 1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa 1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa 1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt 1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc 1441 ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 1501 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc 1561 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 1621 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 1681 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 1741 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 1801 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 1861 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 1921 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 1981 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 2041 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact 2101 ataggggcca ccatggacta taaggaccac gacggagact acaaggatca tgatattgat 2161 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga 2221 gtcccagcag ccgtagattt gagaactttg ggatattcac agcagcagca ggaaaagatc 2281 aagcccaaag tgaggtcgac agtcgcgcag catcacgaag cgctggtggg tcatgggttt 2341 acacatgccc acatcgtagc cttgtcgcag caccctgcag cccttggcac ggtcgccgtc 2401 aagtaccagg acatgattgc ggcgttgccg gaagccacac atgaggcgat cgtcggtgtg 2461 gggaaacagt ggagcggagc ccgagcgctt gaggccctgt tgacggtcgc gggagagctg 2521 agagggcctc cccttcagct ggacacgggc cagttgctga agatcgcgaa gcggggagga 2581 gtcacggcgg tcgaggcggt gcacgcgtgg cgcaatgcgc tcacgggagc acccctcaac 2641 ctgaccccag agcaggtcgt ggcaattgcg agcaacaagg ggggaaagca ggcactcgaa 2701 accgtccaga ggttgctgcc tgtgctgtgc caagcgcacg gacttacgcc agagcaggtc 2761 gtggcaattg cgagcaacgg agggggaaag caggcactcg aaaccgtcca gaggttgctg 2821 cctgtgctgt gccaagcgca cggactaacc ccagagcagg tcgtggcaat tgcgagccat 2881 gacgggggaa agcaggcact cgaaaccgtc cagaggttgc tgcctgtgct gtgccaagcg 2941 cacgggttga ccccagagca ggtcgtggca attgcgagcc atgacggggg aaagcaggca 3001 ctcgaaaccg tccagaggtt gctgcctgtg ctgtgccaag cgcacggctt aaccccagag 3061 caggtcgtgg caattgcgag ccatgacggg ggaaagcagg cactcgaaac cgtccagagg 3121 ttgctgcctg tgctgtgcca agcgcacggc ctgaccccag agcaggtcgt ggcaattgcg 3181 agccatgacg ggggaaagca ggcactcgaa accgtccaga ggttgctgcc tgtgctgtgc 3241 caagcgcacg gactgacacc agagcaggtc gtggcaattg cgagcaacgg agggggaaag 3301 caggcactcg aaaccgtcca gaggttgctg cctgtgctgt gccaagcgca cggactcact 3361 ccagagcagg tcgtggcaat tgcgagccat gacgggggaa agcaggcact cgaaaccgtc 3421 cagaggttgc tgcctgtgct gtgccaagcg cacggcctca ccccagagca ggtcgtggca 3481 attgcgagcc atgacggggg aaagcaggca ctcgaaaccg tccagaggtt gctgcctgtg 3541 ctgtgccaag cgcacggatt aaccccagag caggtcgtgg caattgcgag caacatcggg 3601 ggaaagcagg cactcgaaac cgtccagagg ttgctgcctg tgctgtgcca agcgcacgga 3661 cttaccccag agcaggtcgt ggcaattgcg agccatgacg ggggaaagca ggcactcgaa 3721 accgtccaga ggttgctgcc tgtgctgtgc caagcgcacg ggctcacccc agagcaggtc 3781 gtggcaattg cgagccatga cgggggaaag caggcactcg aaaccgtcca gaggttgctg 3841 cctgtgctgt gccaagcgca cggtcttacc ccagagcagg tcgtggcaat tgcgagccat 3901 gacgggggaa agcaggcact cgaaaccgtc cagaggttgc tgcctgtgct gtgccaagcg 3961 cacggcctca ccccagagca ggtcgtggca attgcgagcc atgacggggg aaagcaggca 4021 ctcgaaaccg tccagaggtt gctgcctgtg ctgtgccaag cgcatgggct caccccagag 4081 caggtcgtgg caattgcgag caacatcggg ggaaagcagg cactcgaaac cgtccagagg 4141 ttgctgcctg tgctgtgcca agcgcacggt ctaaccccag agcaggtcgt ggcaattgcg 4201 agccatgacg ggggaaagca ggcactcgaa accgtccaga ggttgctgcc tgtgctgtgc 4261 caagcgcacg gactcacgcc tgagcaggta gtggctattg catccaacat cgggggcaga 4321 cccgcactgg agtcaatcgt ggcccagctt tcgaggccgg accccgcgct ggccgcactc 4381 actaatgatc atcttgtagc gctggcctgc ctcggcggac gacccgcctt ggatgcggtg 4441 aagaaggggc tcccgcacgc gcctgcattg attaagcgga ccaacagaag gattcccgag 4501 aggacatcac atcgagtggc aggttcccaa ctcgtgaaga gtgaacttga ggagaaaaag 4561 tcggagctgc ggcacaaatt gaaatacgta ccgcatgaat acatcgaact tatcgaaatt 4621 gctaggaact cgactcaaga cagaatcctt gagatgaagg taatggagtt ctttatgaag 4681 gtttatggat accgagggaa gcatctcggt ggatcacgaa aacccgacgg agcaatctat 4741 acggtgggga gcccgattga ttacggagtg atcgtcgaca cgaaagccta cagcggtggg 4801 tacaatcttc ccatcgggca ggcagatgag atgcaacgtt atgtcgaaga aaatcagacc 4861 aggaacaaac acatcaatcc aaatgagtgg tggaaagtgt atccttcatc agtgaccgag 4921 tttaagtttt tgtttgtctc tgggcatttc aaaggcaact ataaggccca gctcacacgg 4981 ttgaatcaca ttacgaactg caatggtgcg gttttgtccg tagaggaact gctcattggt 5041 ggagaaatga tcaaagcggg aactctgaca ctggaagaag tcagacgcaa gtttaacaat 5101 ggcgagatca atttccgctc ataaaaaatc agcctcgact gtgccttcta gttgccagcc 5161 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 5221 cctttcctaa taaaatgagg aaattgcatc acaacactca accctatctc ggtctattct 5281 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 5341 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc 5401 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt 5461 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 5521 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg 5581 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct 5641 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca 5701 aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg atgaaaaagc 5761 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg 5821 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc 5881 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt 5941 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca 6001 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc 6061 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg 6121 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat 6181 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa 6241 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt 6301 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg 6361 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg 6421 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc 6481 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg 6541 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg 6601 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg 6661 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag 6721 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg 6781 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 6841 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 6901 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 6961 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 7021 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat 7081 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 7141 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 7201 agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc agcaataaac 7261 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 7321 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 7381 gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 7441 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 7501 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 7561 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 7621 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 7681 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 7741 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 7801 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 7861 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 7921 cggaaatgtt gaatactcat attcttcctt tttcaatatt attgaagcat ttatcagggt 7981 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtc 8041 agtgttacaa ccaattaacc aattctgaac attatcgcga gcccatttat acctgaatat 8101 ggctcataac accccttg //