LOCUS Exported 5838 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION for e. coli expression, Golden Gate Compatible TALEN incorporated with C Terminal 6xHIS Tag, Codon Optimized FokI. ACCESSION . VERSION . KEYWORDS pET-Sangamo-HIS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5838) TITLE Bao Lab Golden Gate plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 5838) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5838 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 925..944 /label=LacI-R /note="LacI, reverse primer" promoter complement(964..1041) /gene="lacI" /label=lacI promoter primer_bind 1247..1266 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 1350..1368 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1369..1393 /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)." RBS 1408..1430 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1443..1508 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1515..1535 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1692..2267) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG TGERIAPGVHCLYRCYSPSLRDLQQLPGVELERRPPKLTRHSQQRLKCSRSRPLFTDPY NCLVRGFGQGRNHILVFDSHRPKRRRVL" primer_bind 2117..2139 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2131..2148 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2132..2148 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 2158..2176 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2224..2244) /label=T3 /note="T3 promoter, forward primer" promoter complement(2224..2242) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2263..2279) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2263..2279) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2276..2298) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2287..2303 /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(2311..2341) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2356..2377 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." CDS 2563..3150 /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 3166..3183 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3236..3254) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3250..3297 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3334..3789 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3421..3440) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3631..3652 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3882..4697) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 4605..4624 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4689..4708 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4819..5407 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5308..5327 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5561..5578 /label=L4440 /note="L4440 vector, forward primer" CDS complement(5719..963) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" ORIGIN 1 cacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc 61 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata 121 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag 181 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat 241 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc 301 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg 361 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat 421 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga 481 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt 541 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag 601 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct 661 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc 721 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa 781 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat 841 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg 901 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt 961 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt 1021 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta 1081 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat 1141 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga 1201 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga 1261 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt 1321 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc 1381 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg 1441 cggactacaa agaccatgac ggtgattata aagatcatga catcgattac aaggatgacg 1501 atgacaagat ggcccccaag aagaagagga aggtgggcat tcaccgcggg gtacctatgg 1561 tggacttgag gacactcggt tattcgcaac agcaacagga gaaaatcaag cctaaggtca 1621 ggagcaccgt cgcgcaacac cacgaggcgc ttgtggggca tggcttcact catgcgcata 1681 ttgtcgcgct ttcacagcac cctgcggcgc ttgggacggt ggctgtcaaa taccaagata 1741 tgattgcggc cctgcccgaa gccacgcacg aggcaattgt aggggtcggt aaacagtggt 1801 cgggagcgcg agcacttgag gcgctgctga ctgtggcggg tgagcttagg gggcctccgc 1861 tccagctcga caccgggcag ctgctgaaga tcgcgaagag agggggagta acagcggtag 1921 aggcagtgca cgcctggcgc aatgcgctca ccggtgcccc cctggagacg ggcgccgcta 1981 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 2041 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 2101 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa 2161 tacgactcac tatagggcga attgggtacc gggccccccc tcgaggtcct ccagcttttg 2221 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt 2281 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 2341 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 2401 tttccaccgg tcgtctccaa cgaccatctg gtggcgttgg catgtcttgg tggacgaccc 2461 gcgctcgatg cagtcaaaaa gggtctgcct catgctcccg cattgatcaa aagaaccaac 2521 cggcggattc ccgagagaac ttcccatcga gtcgcgggat cccagctggt gaagagcgag 2581 ctggaggaga agaagtccga gctgcggcac aagctgaagt acgtgcccca cgagtacatc 2641 gagctgatcg agatcgccag gaacagcacc caggaccgca tcctggagat gaaggtgatg 2701 gagttcttca tgaaggtgta cggctacagg ggaaagcacc tgggcggaag cagaaagcct 2761 gacggcgcca tctatacagt gggcagcccc atcgattacg gcgtgatcgt ggacacaaag 2821 gcctacagcg gcggctacaa tctgcctatc ggccaggccg acgagatgca gagatacgtg 2881 gaggagaacc agacccggaa taagcacatc aaccccaacg agtggtggaa ggtgtaccct 2941 agcagcgtga ccgagttcaa gttcctgttc gtgagcggcc acttcaaggg caactacaag 3001 gcccagctga ccaggctgaa ccacatcacc aactgcaatg gcgccgtgct gagcgtggag 3061 gagctgctga tcggcggcga gatgatcaaa gccggcaccc tgacactgga ggaggtgcgg 3121 cgcaagttca acaacggcga gatcaacttc gcggccgcac tcgagcacca ccaccaccac 3181 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 3241 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 3301 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg 3361 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 3421 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3481 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 3541 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 3601 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 3661 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 3721 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 3781 ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 3841 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc 3901 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc 3961 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg 4021 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca 4081 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc 4141 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca 4201 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gaggcgaaat 4261 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac 4321 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat 4381 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa 4441 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct 4501 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc 4561 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta 4621 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc 4681 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt 4741 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 4801 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 4861 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 4921 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 4981 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 5041 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 5101 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 5161 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 5221 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 5281 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 5341 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 5401 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 5461 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 5521 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 5581 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggaa ggagctgact 5641 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt 5701 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 5761 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt 5821 ttttcttttc accagtga //