LOCUS Exported 7854 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS TALEN1260 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7854) AUTHORS Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR TITLE Targeted gene disruption in somatic zebrafish cells using engineered TALENs. JOURNAL Nat Biotechnol. 2011 Aug 5;29(8):697-8. doi: 10.1038/nbt.1934. PUBMED 21822241 REFERENCE 2 (bases 1 to 7854) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7854 /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" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 907..972 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 979..999 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3115..3702 /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" CDS 3718..3759 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 3769..3786 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3809..3826) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3815..4039 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 4085..4513 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4172..4191) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4382..4403 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4522..4542) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4527..4857 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4708..4843 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4770..4789 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 4905..4952 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 4971..5369 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" polyA_signal 5527..5648 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5564..5583) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5618..5637 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5697..5713) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5697..5713) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5710..5732) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5721..5737 /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(5745..5775) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5790..5811 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5928..5945) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6099..6687) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6179..6198) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6858..7718) /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 7481..7500 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7719..7823) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 901 accatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat 961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gcattcaccg cggggtacct 1021 atggtggact tgaggacact cggttattcg caacagcaac aggagaaaat caagcctaag 1081 gtcaggagca ccgtcgcgca acaccacgag gcgcttgtgg ggcatggctt cactcatgcg 1141 catattgtcg cgctttcaca gcaccctgcg gcgcttggga cggtggctgt caaataccaa 1201 gatatgattg cggccctgcc cgaagccacg cacgaggcaa ttgtaggggt cggtaaacag 1261 tggtcgggag cgcgagcact tgaggcgctg ctgactgtgg cgggtgagct tagggggcct 1321 ccgctccagc tcgacaccgg gcagctgctg aagatcgcga agagaggggg agtaacagcg 1381 gtagaggcag tgcacgcctg gcgcaatgcg ctcaccgggg cccccttgaa cctgacccca 1441 gaccaggtag tcgcaatcgc gtcgaacatt gggggaaagc aagccctgga aaccgtgcaa 1501 aggttgttgc cggtcctttg tcaagaccac ggccttacac cggagcaagt cgtggccatt 1561 gcaagcaaca tcggtggcaa acaggctctt gagacggttc agagacttct cccagttctc 1621 tgtcaagccc acgggctgac tcccgatcaa gttgtagcga ttgcgtcgaa cattggaggg 1681 aaacaagcat tggagactgt ccaacggctc cttcccgtgt tgtgtcaagc ccacggtttg 1741 acgcctgcac aagtggtcgc catcgccaac aacaacggcg gtaagcaggc gctggaaaca 1801 gtacagcgcc tgctgcctgt actgtgccag gatcatggac tgaccccaga ccaggtagtc 1861 gcaatcgcgt cgaacattgg gggaaagcaa gccctggaaa ccgtgcaaag gttgttgccg 1921 gtcctttgtc aagaccacgg ccttacaccg gagcaagtcg tggccattgc aaataataac 1981 ggtggcaaac aggctcttga gacggttcag agacttctcc cagttctctg tcaagcccac 2041 gggctgactc ccgatcaagt tgtagcgatt gcgtcgcatg acggagggaa acaagcattg 2101 gagactgtcc aacggctcct tcccgtgttg tgtcaagccc acggtttgac gcctgcacaa 2161 gtggtcgcca tcgcctccaa tattggcggt aagcaggcgc tggaaacagt acagcgcctg 2221 ctgcctgtac tgtgccagga tcatggactg accccagacc aggtagtcgc aatcgcgaac 2281 aataatgggg gaaagcaagc cctggaaacc gtgcaaaggt tgttgccggt cctttgtcaa 2341 gaccacggcc ttacaccgga gcaagtcgtg gccattgcaa gcaacatcgg tggcaaacag 2401 gctcttgaga cggttcagag acttctccca gttctctgtc aagcccacgg gctgactccc 2461 gatcaagttg tagcgattgc gtcgaacatt ggagggaaac aagcattgga gactgtccaa 2521 cggctccttc ccgtgttgtg tcaagcccac ggtttgacgc ctgcacaagt ggtcgccatc 2581 gcctccaata ttggcggtaa gcaggcgctg gaaacagtac agcgcctgct gcctgtactg 2641 tgccaggatc atggactgac cccagaccag gtagtcgcaa tcgcgtcaca tgacggggga 2701 aagcaagccc tggaaaccgt gcaaaggttg ttgccggtcc tttgtcaaga ccacggcctt 2761 acaccggagc aagtcgtggc cattgcaagc aatgggggtg gcaaacaggc tcttgagacg 2821 gttcagagac ttctcccagt tctctgtcaa gcccacgggc tgacacccga acaggtggtc 2881 gccattgctt cccacgacgg aggacggcca gccttggagt ccatcgtagc ccaattgtcc 2941 aggcccgatc ccgcgttggc tgcgttaacg aatgaccatc tggtggcgtt ggcatgtctt 3001 ggtggacgac ccgcgctcga tgcagtcaaa aagggtctgc ctcatgctcc cgcattgatc 3061 aaaagaacca accggcggat tcccgagaga acttcccatc gagtcgcggg atcccaacta 3121 gtcaaaagtg aactggagga gaagaaatct gaacttcgtc ataaattgaa atatgtgcct 3181 catgaatata ttgaattaat tgaaattgcc agaaattcca ctcaggatag aattcttgaa 3241 atgaaggtaa tggaattttt tatgaaagtt tatggatata gaggtaaaca tttgggtgga 3301 tcaaggaaac cggacggagc aatttatact gtcggatctc ctattgatta cggtgtgatc 3361 gtggatacta aagcttatag cggaggttat aatctgccaa ttggccaagc agatgaaatg 3421 caacgatatg tcgaagaaaa tcaaacacga aacaaacata tcaaccctaa tgaatggtgg 3481 aaagtctatc catcttctgt aacggaattt aagtttttat ttgtgagtgg tcactttaaa 3541 ggaaactaca aagctcagct tacacgatta aatcatatca ctaattgtaa tggagctgtt 3601 cttagtgtag aagagctttt aattggtgga gaaatgatta aagccggcac attaacctta 3661 gaggaagtca gacggaaatt taataacggc gagataaact tttaagggcc cttcgaaggt 3721 aagcctatcc ctaaccctct cctcggtctc gattctacgc gtaccggtca tcatcaccat 3781 caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 3841 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 3901 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 3961 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 4021 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 4081 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 4141 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 4201 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt agggttccga 4261 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 4321 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 4381 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 4441 ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 4501 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 4561 ccccaggcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga 4621 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 4681 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat 4741 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc 4801 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag 4861 ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat 4921 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagc 4981 ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc 5041 ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca 5101 ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca 5161 ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca 5221 ggggcatctt gagcccctgc ggacggtgtc gacaggtgct tctcgatctg catcctggga 5281 tcaaagcgat agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc 5341 tgccctctgg ttatgtgtgg gagggctaag cacttcgtgg ccgaggagca ggactgacac 5401 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 5461 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 5521 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 5581 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 5641 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca 5701 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 5761 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 5821 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5881 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 5941 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 6001 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 6061 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 6121 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 6181 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 6241 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca 6301 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 6361 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 6421 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 6481 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 6541 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 6601 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 6661 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 6721 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 6781 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6841 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6901 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6961 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 7021 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 7081 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 7141 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 7201 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 7261 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 7321 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 7381 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 7441 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 7501 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 7561 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 7621 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 7681 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 7741 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 7801 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc //