LOCUS Exported 7048 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of Zdk1 tagged VAV2 DH/PH/C1 domain. ACCESSION . VERSION . KEYWORDS pTriEx-mVenus-Zdk1-VAV2 DH/PH/C1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7048) 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 7048) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7048 /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..2759 /codon_start=1 /product="photostable derivative of mVenus (Lee et al., 2020)" /label=mGold /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFISTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2088..2109) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2349..2368) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2696..2717 /label=EGFP-C /note="EGFP, forward primer" CDS 2778..2954 /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 4191..4223 /codon_start=1 /product="HSV (herpes simplex virus) epitope tag" /label=HSV tag /translation="QPELAPEDPED" CDS 4230..4253 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" primer_bind complement(4343..4362) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 4408..4463 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(4462..4481) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" terminator 4551..4598 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" misc_recomb 4611..5316 /label=baculovirus recombination region (ORF1629) /note="contains part of ORF1629" 3'UTR 4699..5071 /label=polyhedrin 3' UTR /note="3' UTR from the Autographa californica nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)" CDS complement(4728..5316) /codon_start=2 /product="baculovirus capsid-associated protein" /label=ORF1629 /note="required for viral replication" /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA" protein_bind 5325..5358 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." rep_origin complement(5526..6113) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5605..5624) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6126..6986) /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 6749..6768 /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 gagctgttca ccggggtggt gcccatcctg gtcgagctgg 2101 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 2161 acggcaagct gaccctgaag ttcatcagca ccaccggcaa gctgcccgtg ccctggccca 2221 ccctcgtgac caccctcggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga 2281 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 2341 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 2401 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 2461 acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga 2521 acggcatcaa ggcaaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg 2581 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 2641 actacctgag ctaccagtcc aaactgagca aagaccccaa cgagaagcgc gatcacatgg 2701 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg 2761 gttctggaag tggatctatg gtggataaca aattcaataa agaaaagacg cgtgccggtg 2821 cggaaatcca ttctctgcca aacctgaatg ttgagcagaa gtttgccttc atcgtgagcc 2881 tgtttgatga tccatctcag agcgccaatc tgctggccga agccaaaaaa ctgaacgatg 2941 cccaggcccc aaaaggatcc ggatctggtg gatctggtaa aatgggcatg actgaggacg 3001 acaagagaag ctgctgcttg ttagagattc aggagaccga ggccaagtac taccgcaccc 3061 tggaggacat tgagaagaac tacatgggtc ccttgcggct ggtgctgagc ccggcggata 3121 tggctgctgt cttcatcaac ctggaggacc tcatcaaggt gcatcacagc tttctgcgag 3181 ccatcgatgt gtccatgatg gctggtggca gtaccctggc taaggtcttt ctggagttta 3241 aggaaaggct cctgatctat ggagagtact gtagccacat ggaacacgct cagagtacac 3301 tgaaccagct cctcgccagc cgagaggact tcaggcagaa agtggaggag tgcacactca 3361 gggttcagga tggcaagttc aagctgcaag acctgctggt ggtgcccatg caacgggtgc 3421 tgaagtacca cctgctgctc aaggagctcc tgagccattc tgcagaccga ccagaaagac 3481 aacagctcaa agaagccctg gaagccatgc aggacttggc catgtacatc aatgaagtga 3541 agcgggacaa ggagaccttg aagaagatta gcgagttcca gtgctccata gaaaacctgc 3601 aagtgaagct ggaggaattt gggaggccaa agattgacgg ggagcttaaa gtccggtcca 3661 tagtcaacca caccaagcaa gacaggtacc tgttcctatt tgacaaggtg gtcatcgtgt 3721 gcaagaggaa gggctacagc tatgagctga aggaggtcat tgagctgctc ttccacaaga 3781 tgaccgatga cccgatgcac aacaaggaca tcaagaagtg gtcctatggc ttctacctga 3841 ttcacctcca aggaaagcaa ggctttcagt tcttctgcaa gacggaagac atgaagcgga 3901 agtggatgga gcagttcgag atggccatgt caaacatcaa gccagataag gccaatgcca 3961 accatcatag cttccagatg tacacattcg acaagactac caactgcaaa gcctgcaaga 4021 tgtttctcag gggtaccttc taccagggat acctgtgtac cagatgtggc gtcggggcac 4081 acaaggaatg cctggaggtg atccccccct gcaagatgag ttcacctgca gatgtggatg 4141 ctcctggagc ataaaagctt gcggccgcac agctgtatac acgtgcaagc cagccagaac 4201 tcgccccgga agaccccgag gatctcgagc accaccatca ccatcaccat cactaagtga 4261 ttaacctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc 4321 tcacaaatac cactgagatc gatctttttc cctctgccaa aaattatggg gacatcatga 4381 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 4441 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 4501 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgtaa ctagcataac 4561 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaagcatgc ggaggaaatt 4621 ctccttgaag tttccctggt gttcaaagta aaggagtttg caccagacgc acctctgttc 4681 actggtccgg cgtattaaaa cacgatacat tgttattagt acatttatta agcgctagat 4741 tctgtgcgtt gttgatttac agacaattgt tgtacgtatt ttaataattc attaaattta 4801 taatctttag ggtggtatgt tagagcgaaa atcaaatgat tttcagcgtc tttatatctg 4861 aatttaaata ttaaatcctc aatagatttg taaaataggt ttcgattagt ttcaaacaag 4921 ggttgttttt ccgaaccgat ggctggacta tctaatggat tttcgctcaa cgccacaaaa 4981 cttgccaaat cttgtagcag caatctagct ttgtcgatat tcgtttgtgt tttgttttgt 5041 aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg tatttctttc atcactgtcg 5101 ttagtgtaca attgactcga cgtaaacacg ttaaatagag cttggacata tttaacatcg 5161 ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc aaccctcgtc gttagaagtt 5221 gcttccgaag acgattttgc catagccaca cgacgcctat taattgtgtc ggctaacacg 5281 tccgcgatca aatttgtagt tgagcttttt ggaattgcga tcgcataact tcgtatagca 5341 tacattatac gaagttataa gctcggaacg ctgcgctcgg tcgttcggct gcggcgagcg 5401 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 5461 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 5521 gcgtttttcc ataggctccg ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5581 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5641 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5701 gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc 5761 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5821 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5881 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5941 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 6001 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 6061 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 6121 ctttgttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 6181 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 6241 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 6301 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 6361 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 6421 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 6481 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 6541 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 6601 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 6661 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 6721 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 6781 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 6841 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 6901 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 6961 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 7021 cagggttatt gtctcatgtc cgcgcgtt //