LOCUS Exported 5768 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of TALEN proteins with FokI-DDD domain. ACCESSION . VERSION . KEYWORDS pCS2TAL3-DDD SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5768) AUTHORS Hu R, Wallace J, Dahlem TJ, Grunwald DJ, O'Connell RM TITLE Targeting human microRNA genes using engineered Tal-effector nucleases (TALENs). JOURNAL PLoS One. 2013 May 7;8(5):e63074. doi: 10.1371/journal.pone.0063074. Print 2013. PUBMED 23667577 REFERENCE 2 (bases 1 to 5768) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5768 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 35..53 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 35..52 /label=SP6 /note="SP6 promoter, forward primer" regulatory 85..94 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 97..117 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 118..183 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS complement(490..897) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG TRQCIARCMHCLHCGHAATFCNLHKLACVQL" primer_bind 747..769 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 761..778 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 762..778 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 788..807 /label=T7 /note="T7 promoter, forward primer" promoter 788..806 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter complement(854..872) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(893..909) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 917..933 /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(941..971) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 986..1007 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." CDS 1193..1780 /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 MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQD YVEENQTRDKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF" promoter complement(1791..1808) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 1813..1947 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1837..1856) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1891..1910 /label=SV40pA-R /note="SV40 polyA, reverse primer" promoter complement(2064..2082) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2103..2119) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2127..2143 /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(2151..2181) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2196..2217 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2334..2351) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2505..3093) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2585..2604) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3264..4124) /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 3887..3906 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4125..4229) /gene="bla" /label=AmpR promoter rep_origin complement(4255..4710) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4392..4413) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4604..4623 /label=F1ori-R /note="F1 origin, reverse primer" promoter 4784..5768 /label=CMV IE94 promoter /note="enhancer/promoter region of simian cytomegalovirus major immediate early transcription unit IE94" ORIGIN 1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag 61 ctacttgttc tttttgcagg atctgccacc atggctccaa agaagaagcg taaggtagac 121 tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac 181 aagggtaccg tggatctacg cacgctcggc tacagtcagc agcagcaaga gaagatcaaa 241 ccgaaggtgc gttcgacagt ggcgcagcac cacgaggcac tggtgggcca tgggtttaca 301 cacgcgcaca tcgttgcgct cagccaacac ccggcagcgt tagggaccgt cgctgtcacg 361 tatcagcaca taatcacggc gttgccagag gcgacacacg aagacatcgt tggcgtcggc 421 aaacagtggt ccggcgcacg cgccctggag gccttgctca cggatgcggg ggagttgaga 481 ggtccgccgt tacagttgga cacaggccaa cttgtgaaga ttgcaaaacg tggcggcgtg 541 accgcaatgg aggcagtgca tgcatcgcgc aatgcactga cgggtgcccc cctggagacg 601 ggcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg 661 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg 721 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga 781 gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc tcgaggtcct 841 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 901 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 961 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 1021 cactgcccgc tttccaccgg tcgtctccaa cgaccacctc gtcgccttgg cctgcctcgg 1081 cggacgtcct gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag 1141 aagagtcaat cgccgtattg gcgaacgcac gtcccatcgc gttgccggat cccaactagt 1201 caaaagtgaa ctggaggaga agaaatctga acttcgtcat aaattgaaat atgtgcctca 1261 tgaatatatt gaattaattg aaattgccag aaattccact caggatagaa ttcttgaaat 1321 gaaggtaatg gaatttttta tgaaagttta tggatataga ggtaaacatt tgggtggatc 1381 aaggaaaccg gacggagcaa tttatactgt cggatctcct attgattacg gtgtgatcgt 1441 ggatactaaa gcttatagcg gaggttataa tctgccaatt ggccaagcag atgaaatgca 1501 agactatgtc gaagaaaatc aaacacgaga caaacatatc aaccctaatg aatggtggaa 1561 agtctatcca tcttctgtaa cggaatttaa gtttttattt gtgagtggtc actttaaagg 1621 aaactacaaa gctcagctta cacgattaaa tcatatcact aattgtaatg gagctgttct 1681 tagtgtagaa gagcttttaa ttggtggaga aatgattaaa gccggcacat taaccttaga 1741 ggaagtgcgc cggaaattta ataacggcga gataaacttt taatctagaa ctatagtgag 1801 tcgtattacg tagatccaga catgataaga tacattgatg agtttggaca aaccacaact 1861 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 1921 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag 1981 gttcaggggg aggtgtggga ggttttttaa ttcgcggccg cggcgccaat gcattgggcc 2041 cggtacgtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca 2101 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 2161 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 2221 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 2281 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 2341 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 2401 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 2461 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 2521 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 2581 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 2641 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 2701 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 2761 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 2821 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 2881 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 2941 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 3001 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 3061 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3121 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 3181 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 3241 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 3301 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 3361 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 3421 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 3481 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 3541 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 3601 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 3661 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 3721 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 3781 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 3841 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 3901 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 3961 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 4021 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 4081 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 4141 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 4201 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg 4261 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 4321 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 4381 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 4441 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 4501 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat 4561 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag 4621 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg 4681 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt 4741 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccata gccaattcaa 4801 tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt gccaattcaa 4861 tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca gccaattcaa 4921 tatggcggac ttggcaccat gccaattcaa tatggcggac ttggcactgt gccaactggg 4981 gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg tgccaagtcc 5041 gccatattga attggcatgg tgccaataat ggcggccata ttggctatat gccaggatca 5101 atatataggc aatatccaat atggccctat gccaatatgg ctattggcca ggttcaatac 5161 tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt gacgtagata 5221 gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac cgcccatagc 5281 cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac gtcatatggg 5341 cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta aatggcccgc 5401 ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa tgggatggct 5461 cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg taaatggccc 5521 acttggcagt acatcaatat ctattaatag taacttggca agtacattac tattggaagg 5581 acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg gcccgcgatg 5641 gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa atgggcgttc 5701 cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc aatgctcgtt 5761 tagggaac //