LOCUS Exported 6447 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS JDS74 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6447) 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 6447) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6447 /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 1708..2295 /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 2311..2352 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 2362..2379 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2402..2419) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2408..2632 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2678..3106 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2765..2784) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2975..2996 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3115..3135) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3120..3450 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3301..3436 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3363..3382 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 3498..3545 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 3564..3962 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" polyA_signal 4120..4241 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4157..4176) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4211..4230 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4290..4306) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4290..4306) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4303..4325) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4314..4330 /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(4338..4368) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4383..4404 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4521..4538) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4692..5280) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4772..4791) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5451..6311) /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 6074..6093 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6312..6416) /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 cagagacgat 1441 taatgcgtct cgctgacacc cgaacaggtg gtcgccattg ctaataataa cggaggacgg 1501 ccagccttgg agtccatcgt agcccaattg tccaggcccg atcccgcgtt ggctgcgtta 1561 acgaatgacc atctggtggc gttggcatgt cttggtggac gacccgcgct cgatgcagtc 1621 aaaaagggtc tgcctcatgc tcccgcattg atcaaaagaa ccaaccggcg gattcccgag 1681 agaacttccc atcgagtcgc gggatcccaa ctagtcaaaa gtgaactgga ggagaagaaa 1741 tctgaacttc gtcataaatt gaaatatgtg cctcatgaat atattgaatt aattgaaatt 1801 gccagaaatt ccactcagga tagaattctt gaaatgaagg taatggaatt ttttatgaaa 1861 gtttatggat atagaggtaa acatttgggt ggatcaagga aaccggacgg agcaatttat 1921 actgtcggat ctcctattga ttacggtgtg atcgtggata ctaaagctta tagcggaggt 1981 tataatctgc caattggcca agcagatgaa atgcaacgat atgtcgaaga aaatcaaaca 2041 cgaaacaaac atatcaaccc taatgaatgg tggaaagtct atccatcttc tgtaacggaa 2101 tttaagtttt tatttgtgag tggtcacttt aaaggaaact acaaagctca gcttacacga 2161 ttaaatcata tcactaattg taatggagct gttcttagtg tagaagagct tttaattggt 2221 ggagaaatga ttaaagccgg cacattaacc ttagaggaag tcagacggaa atttaataac 2281 ggcgagataa acttttaagg gcccttcgaa ggtaagccta tccctaaccc tctcctcggt 2341 ctcgattcta cgcgtaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca 2401 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 2461 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 2521 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 2581 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 2641 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 2701 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 2761 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 2821 gctctaaatc ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc 2881 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 2941 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 3001 acactcaacc ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc 3061 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 3121 tgtgtcagtt agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc 3181 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga 3241 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc 3301 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 3361 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga 3421 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc 3481 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat 3541 acgacaaggt gaggaactaa accatggcca agcctttgtc tcaagaagaa tccaccctca 3601 ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca 3661 gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg 3721 ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc 3781 tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt 3841 gtcgacaggt gcttctcgat ctgcatcctg ggatcaaagc gatagtgaag gacagtgatg 3901 gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct 3961 aagcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc 4021 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 4081 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat 4141 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 4201 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg 4261 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 4321 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 4381 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 4441 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 4501 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 4561 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4621 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4681 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4741 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4801 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4861 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag 4921 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 4981 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 5041 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 5101 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 5161 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 5221 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 5281 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 5341 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 5401 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 5461 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 5521 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 5581 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 5641 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 5701 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 5761 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 5821 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 5881 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 5941 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 6001 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 6061 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 6121 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 6181 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 6241 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 6301 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 6361 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 6421 tttccccgaa aagtgccacc tgacgtc //