LOCUS Exported 5929 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of Zdk1. ACCESSION . VERSION . KEYWORDS pTriEx-mCherry-Zdk1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5929) AUTHORS Wang H, Vilela M, Winkler A, Tarnawski M, Schlichting I, Yumerefendi H, Kuhlman B, Liu R, Danuser G, Hahn KM TITLE LOVTRAP: an optogenetic system for photoinduced protein dissociation. JOURNAL Nat Methods. 2016 Jul 18. doi: 10.1038/nmeth.3926. PUBMED 27427858 REFERENCE 2 (bases 1 to 5929) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5929 /organism="synthetic DNA construct" /mol_type="other DNA" misc_recomb 110..937 /label=baculovirus recombination region (lef2/ORF603) /note="contains ORF603 and part of lef2" CDS 110..234 /codon_start=3 /product="baculovirus late expression factor 2" /label=lef2 /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY" CDS complement(272..877) /codon_start=1 /product="baculovirus ORF603 protein" /label=ORF603 /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR" enhancer 1005..1308 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1310..1509 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1463..1483 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 1715..1737 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1799..1818 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" primer_bind 1860..1879 /label=T7 /note="T7 promoter, forward primer" promoter 1860..1878 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1879..1903 /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 1919..2028 /label=p10 promoter /note="baculovirus promoter for expression in insect cells" CDS 2043..2750 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(2188..2206) /label=mCherry-R /note="mCherry, reverse primer" CDS 2763..2939 /codon_start=1 /product="""Zdark"" protein, derived from the Z subunit of protein A, that binds to the dark state of LOV2 and is released upon irradiation (Wang et al., 2016)" /label=Zdk1 /translation="MVDNKFNKEKTRAGAEIHSLPNLNVEQKFAFIVSLFDDPSQSANL LAEAKKLNDAQAPK" CDS 2958..2975 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 2982..3002 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3009..3032 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 3072..3104 /codon_start=1 /product="HSV (herpes simplex virus) epitope tag" /label=HSV tag /translation="QPELAPEDPED" CDS 3111..3134 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" primer_bind complement(3224..3243) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 3289..3344 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(3343..3362) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" terminator 3432..3479 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" misc_recomb 3492..4197 /label=baculovirus recombination region (ORF1629) /note="contains part of ORF1629" 3'UTR 3580..3952 /label=polyhedrin 3' UTR /note="3' UTR from the Autographa californica nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)" CDS complement(3609..4197) /codon_start=2 /product="baculovirus capsid-associated protein" /label=ORF1629 /note="required for viral replication" /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA" protein_bind 4206..4239 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." rep_origin complement(4407..4994) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4486..4505) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5007..5867) /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 5630..5649 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 aactgttaca ttgcaaacgt ggtttcgtgt gccaagtgtg aaaaccgatg tttaatcaag 61 gctctgacgc atttctacaa ccacgactcc aagtgtgtgg gtgaagtcat gcatctttta 121 atcaaatccc aagatgtgta taaaccacca aactgccaaa aaatgaaaac tgtcgacaag 181 ctctgtccgt ttgctggcaa ctgcaagggt ctcaatccta tttgtaatta ttgaataata 241 aaacaattat aaatgtcaaa tttgtttttt attaacgata caaaccaaac gcaacaagaa 301 catttgtagt attatctata attgaaaacg cgtagttata atcgctgagg taatatttaa 361 aatcattttc aaatgattca cagttaattt gcgacaatat aattttattt tcacataaac 421 tagacgcctt gtcgtcttct tcttcgtatt ccttctcttt ttcatttttc tcttcataaa 481 aattaacata gttattatcg tatccatata tgtatctatc gtatagagta aattttttgt 541 tgtcataaat atatatgtct tttttaatgg ggtgtatagt accgctgcgc atagtttttc 601 tgtaatttac aacagtgcta ttttctggta gttcttcgga gtgtgttgct ttaattatta 661 aatttatata atcaatgaat ttgggatcgt cggttttgta caatatgttg ccggcatagt 721 acgcagcttc ttctagttca attacaccat tttttagcag caccggatta acataacttt 781 ccaaaatgtt gtacgaaccg ttaaacaaaa acagttcacc tcccttttct atactattgt 841 ctgcgagcag ttgtttgttg ttaaaaataa cagccattgt aatgagacgc acaaactaat 901 atcacaaact ggaaatgtct atcaatatat agttgctgat ggccggccta ttaatagtaa 961 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 1021 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 1081 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 1141 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt 1201 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac 1261 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt 1321 tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 1381 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 1441 cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 1501 ataagcagac gtcgtttagt gaaccgtcag atcactagat gctttattgc ggtagtttat 1561 cacagttaaa ttgctaacgc cagtctcgaa cttaacgtgc agaagttggt cgtgaggcac 1621 tgggcaggta agtatcgggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg 1681 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 1741 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 1801 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcg gaccgaaatt 1861 aatacgactc actatagggg aattgtgagc ggataacaat tccccggagt taatccggga 1921 cctttaattc aacccaacac aatatattat agttaaataa gaattattat caaatcattt 1981 gtatattaat taaaatacta tactgtaaat tacattttat ttacaatcaa aggagatata 2041 ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca 2101 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg 2161 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc 2221 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc 2281 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc 2341 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg 2401 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccctg 2461 taatgcagaa gaagactatg ggctgggagg cctcctccga gcggatgtac cccgaggacg 2521 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg 2581 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg 2641 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg 2701 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag ggttctggat 2761 ccatggtgga taacaaattc aataaagaaa agacgcgtgc cggtgcggaa atccattctc 2821 tgccaaacct gaatgttgag cagaagtttg ccttcatcgt gagcctgttt gatgatccat 2881 ctcagagcgc caatctgctg gccgaagcca aaaaactgaa cgatgcccag gccccaaaag 2941 gtggttctgg tggtagccat catcatcatc atcatggcgg tgaaaatctg tatttccagg 3001 gcggccgcga ctacaaggac gacgacgaca aataaaagct tgcggccgca cagctgtata 3061 cacgtgcaag ccagccagaa ctcgccccgg aagaccccga ggatctcgag caccaccatc 3121 accatcacca tcactaagtg attaacctca ggtgcaggct gcctatcaga aggtggtggc 3181 tggtgtggcc aatgccctgg ctcacaaata ccactgagat cgatcttttt ccctctgcca 3241 aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat 3301 ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg 3361 ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg 3421 cccatatgta actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc 3481 tgaaagcatg cggaggaaat tctccttgaa gtttccctgg tgttcaaagt aaaggagttt 3541 gcaccagacg cacctctgtt cactggtccg gcgtattaaa acacgataca ttgttattag 3601 tacatttatt aagcgctaga ttctgtgcgt tgttgattta cagacaattg ttgtacgtat 3661 tttaataatt cattaaattt ataatcttta gggtggtatg ttagagcgaa aatcaaatga 3721 ttttcagcgt ctttatatct gaatttaaat attaaatcct caatagattt gtaaaatagg 3781 tttcgattag tttcaaacaa gggttgtttt tccgaaccga tggctggact atctaatgga 3841 ttttcgctca acgccacaaa acttgccaaa tcttgtagca gcaatctagc tttgtcgata 3901 ttcgtttgtg ttttgttttg taataaaggt tcgacgtcgt tcaaaatatt atgcgctttt 3961 gtatttcttt catcactgtc gttagtgtac aattgactcg acgtaaacac gttaaataga 4021 gcttggacat atttaacatc gggcgtgtta gctttattag gccgattatc gtcgtcgtcc 4081 caaccctcgt cgttagaagt tgcttccgaa gacgattttg ccatagccac acgacgccta 4141 ttaattgtgt cggctaacac gtccgcgatc aaatttgtag ttgagctttt tggaattgcg 4201 atcgcataac ttcgtatagc atacattata cgaagttata agctcggaac gctgcgctcg 4261 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 4321 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 4381 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gccccctgac gagcatcaca 4441 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4501 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4561 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 4621 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4681 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 4741 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 4801 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 4861 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 4921 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 4981 aaaaaggatc tcaagaagat cctttgttac caatgcttaa tcagtgaggc acctatctca 5041 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 5101 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 5161 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 5221 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 5281 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 5341 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 5401 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 5461 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 5521 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 5581 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 5641 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 5701 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 5761 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 5821 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 5881 caatattatt gaagcattta tcagggttat tgtctcatgt ccgcgcgtt //