LOCUS Exported 8058 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Park2_L (TAL 3012) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8058) 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 8058) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8058 /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 3319..3906 /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 3922..3963 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 3973..3990 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4013..4030) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4019..4243 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 4289..4717 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4376..4395) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4586..4607 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4726..4746) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4731..5061 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4912..5047 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4974..4993 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 5109..5156 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 5175..5573 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" polyA_signal 5731..5852 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5768..5787) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5822..5841 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5901..5917) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5901..5917) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5914..5936) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5925..5941 /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(5949..5979) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5994..6015 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6132..6149) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6303..6891) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6383..6402) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7062..7922) /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 7685..7704 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7923..8027) /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 gaacaataat gggggaaagc aagccctgga aaccgtgcaa 1501 aggttgttgc cggtcctttg tcaagaccac ggccttacac cggagcaagt cgtggccatt 1561 gcaaataata acggtggcaa 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 cacatgacgg gggaaagcaa gccctggaaa ccgtgcaaag gttgttgccg 1921 gtcctttgtc aagaccacgg ccttacaccg gagcaagtcg tggccattgc aagcaacatc 1981 ggtggcaaac aggctcttga gacggttcag agacttctcc cagttctctg tcaagcccac 2041 gggctgactc ccgatcaagt tgtagcgatt gcgaataaca atggagggaa acaagcattg 2101 gagactgtcc aacggctcct tcccgtgttg tgtcaagccc acggtttgac gcctgcacaa 2161 gtggtcgcca tcgccaacaa caacggcggt aagcaggcgc tggaaacagt acagcgcctg 2221 ctgcctgtac tgtgccagga tcatggactg accccagacc aggtagtcgc aatcgcgaac 2281 aataatgggg gaaagcaagc cctggaaacc gtgcaaaggt tgttgccggt cctttgtcaa 2341 gaccacggcc ttacaccgga gcaagtcgtg gccattgcaa gcaatggggg tggcaaacag 2401 gctcttgaga cggttcagag acttctccca gttctctgtc aagcccacgg gctgactccc 2461 gatcaagttg tagcgattgc gaataacaat ggagggaaac aagcattgga gactgtccaa 2521 cggctccttc ccgtgttgtg tcaagcccac ggtttgacgc ctgcacaagt ggtcgccatc 2581 gccagccatg atggcggtaa gcaggcgctg gaaacagtac agcgcctgct gcctgtactg 2641 tgccaggatc atggactgac cccagaccag gtagtcgcaa tcgcgaacaa taatggggga 2701 aagcaagccc tggaaaccgt gcaaaggttg ttgccggtcc tttgtcaaga ccacggcctt 2761 acaccggagc aagtcgtggc cattgcaagc aacatcggtg gcaaacaggc tcttgagacg 2821 gttcagagac ttctcccagt tctctgtcaa gcccacgggc tgactcccga tcaagttgta 2881 gcgattgcga ataacaatgg agggaaacaa gcattggaga ctgtccaacg gctccttccc 2941 gtgttgtgtc aagcccacgg tttgacgcct gcacaagtgg tcgccatcgc ctcgaatggc 3001 ggcggtaagc aggcgctgga aacagtacag cgcctgctgc ctgtactgtg ccaggatcat 3061 ggactgacac ccgaacaggt ggtcgccatt gctaataata acggaggacg gccagccttg 3121 gagtccatcg tagcccaatt gtccaggccc gatcccgcgt tggctgcgtt aacgaatgac 3181 catctggtgg cgttggcatg tcttggtgga cgacccgcgc tcgatgcagt caaaaagggt 3241 ctgcctcatg ctcccgcatt gatcaaaaga accaaccggc ggattcccga gagaacttcc 3301 catcgagtcg cgggatccca actagtcaaa agtgaactgg aggagaagaa atctgaactt 3361 cgtcataaat tgaaatatgt gcctcatgaa tatattgaat taattgaaat tgccagaaat 3421 tccactcagg atagaattct tgaaatgaag gtaatggaat tttttatgaa agtttatgga 3481 tatagaggta aacatttggg tggatcaagg aaaccggacg gagcaattta tactgtcgga 3541 tctcctattg attacggtgt gatcgtggat actaaagctt atagcggagg ttataatctg 3601 ccaattggcc aagcagatga aatgcaacga tatgtcgaag aaaatcaaac acgaaacaaa 3661 catatcaacc ctaatgaatg gtggaaagtc tatccatctt ctgtaacgga atttaagttt 3721 ttatttgtga gtggtcactt taaaggaaac tacaaagctc agcttacacg attaaatcat 3781 atcactaatt gtaatggagc tgttcttagt gtagaagagc ttttaattgg tggagaaatg 3841 attaaagccg gcacattaac cttagaggaa gtgagacgga aatttaataa cggcgagata 3901 aacttttaag ggcccttcga aggtaagcct atccctaacc ctctcctcgg tctcgattct 3961 acgcgtaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact 4021 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 4081 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 4141 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 4201 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 4261 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg 4321 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 4381 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 4441 cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 4501 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 4561 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 4621 cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc ctattggtta 4681 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt 4741 tagggtgtgg aaagtcccca ggctccccag gcaggcagaa gtatgcaaag catgcatctc 4801 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 4861 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 4921 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 4981 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 5041 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 5101 cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg 5161 tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag 5221 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc 5281 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt 5341 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta 5401 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgtcgacagg 5461 tgcttctcga tctgcatcct gggatcaaag cgatagtgaa ggacagtgat ggacagccga 5521 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacttc 5581 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat 5641 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 5701 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 5761 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 5821 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 5881 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 5941 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 6001 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 6061 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 6121 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 6181 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 6241 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 6301 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 6361 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 6421 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 6481 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 6541 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 6601 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 6661 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 6721 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 6781 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 6841 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 6901 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 6961 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 7021 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 7081 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 7141 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 7201 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 7261 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 7321 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 7381 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 7441 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 7501 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 7561 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 7621 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 7681 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 7741 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 7801 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 7861 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 7921 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 7981 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 8041 aaagtgccac ctgacgtc //