LOCUS Exported 7742 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses CRY2 (full length, intact NLS) fusion with Gal4DBD(aa1-147) fused to VP16AD (truncated), downstream of mCherry-IRES.. ACCESSION . VERSION . KEYWORDS CRY-GalVP16 (B695) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7742) AUTHORS Pathak GP, Spiltoir JI, Hoglund C, Polstein LR, Heine-Koskinen S, Gersbach CA, Rossi J, Tucker CL TITLE Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2. JOURNAL Nucleic Acids Res. 2017 Apr 20. doi: 10.1093/nar/gkx260. PUBMED 28431041 REFERENCE 2 (bases 1 to 7742) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7742 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 61..364 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 365..568 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 519..539 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 607..616 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 613..1320 /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(758..776) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1026..1045 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(1922..1938) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1923..1939) /label=pBluescriptKS /note="For pBluescript vector" CDS 1950..3785 /codon_start=1 /product="Arabidopsis thaliana cryptochrome 2" /label=CRY2 /note="binds to CIB1 upon exposure to blue light (Kennedy et al., 2010)" /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKE PGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSV FFVSQSCSLASEGKNLEGIQDSSDQITTSLGKNGCK" CDS 3831..4271 /codon_start=1 /gene="Saccharomyces cerevisiae GAL4 (truncated)" /product="DNA binding domain of the GAL4 transcriptional activator" /label=GAL4 DNA binding domain /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS" polyA_signal 4530..4651 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4567..4586) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4621..4640 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(4658..5113) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4795..4816) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5007..5026 /label=F1ori-R /note="F1 origin, reverse primer" promoter 5140..5244 /gene="bla" /label=AmpR promoter promoter 5246..5603 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(5269..5289) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 5454..5589 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5516..5535 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5638..6432 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(5692..5711) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6302..6321 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(6620..6639) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 6664..6711 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 7040..7628 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7529..7548 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 601 ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc 661 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc 721 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt 781 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc 841 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc 901 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc 961 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc 1021 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac 1081 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc 1141 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc 1201 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg 1261 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag 1321 tgtggcggct agtactccgg tattgcggta cccttgtacg cctgttttat actcccttcc 1381 cgtaacttag acgcacaaaa ccaagttcaa tagaaggggg tacaaaccag taccaccacg 1441 aacaagcact tctgtttccc cggtgatgtc gtatagactg cttgcgtggt tgaaagcgac 1501 ggatccgtta tccgcttatg tacttcgaga agcccagtac cacctcggaa tcttcgatgc 1561 gttgcgctca gcactcaacc ccagagtgta gcttaggctg atgagtctgg acatccctca 1621 ccggtgacgg tggtccaggc tgcgttggcg gcctacctat ggctaacgcc atgggacgct 1681 agttgtgaac aaggtgtgaa gagcctattg agctacataa gaatcctccg gcccctgaat 1741 gcggctaatc ccaacctcgg agcaggtggt cacaaaccag tgattggcct gtcgtaacgc 1801 gcaagtccgt ggcggaaccg actactttgg gtgtccgtgt ttccttttat tttattgtgg 1861 ctgcttatgg tgacaatcac agattgttat cataaagcga attggatagg atcaagctta 1921 tcgataccgt cgacctcgag ctcgccacca tgaagatgga caaaaagact atagtttggt 1981 ttagaagaga cctaaggatt gaggataatc ctgcattagc agcagctgct cacgaaggat 2041 ctgtttttcc tgtcttcatt tggtgtcctg aagaagaagg acagttttat cctggaagag 2101 cttcaagatg gtggatgaaa caatcacttg ctcacttatc tcaatccttg aaggctcttg 2161 gatctgacct cactttaatc aaaacccaca acacgatttc agcgatcttg gattgtatcc 2221 gcgttaccgg tgctacaaaa gtcgtcttta accacctcta tgatcctgtt tcgttagttc 2281 gggaccatac cgtaaaggag aagctggtgg aacgtgggat ctctgtgcaa agctacaatg 2341 gagatctatt gtatgaaccg tgggagatat actgcgaaaa gggcaaacct tttacgagtt 2401 tcaattctta ctggaagaaa tgcttagata tgtcgattga atccgttatg cttcctcctc 2461 cttggcggtt gatgccaata actgcagcgg ctgaagcgat ttgggcgtgt tcgattgaag 2521 aactagggct ggagaatgag gccgagaaac cgagcaatgc gttgttaact agagcttggt 2581 ctccaggatg gagcaatgct gataagttac taaatgagtt catcgagaag cagttgatag 2641 attatgcaaa gaacagcaag aaagttgttg ggaattctac ttcactactt tctccgtatc 2701 tccatttcgg ggaaataagc gtcagacacg ttttccagtg tgcccggatg aaacaaatta 2761 tatgggcaag agataagaac agtgaaggag aagaaagtgc agatcttttt cttaggggaa 2821 tcggtttaag agagtattct cggtatatat gtttcaactt cccgtttact cacgagcaat 2881 cgttgttgag tcatcttcgg tttttccctt gggatgctga tgttgataag ttcaaggcct 2941 ggagacaagg caggaccggt tatccgttgg tggatgccgg aatgagagag ctttgggcta 3001 ccggatggat gcataacaga ataagagtga ttgtttcaag ctttgctgtg aagtttcttc 3061 tccttccatg gaaatgggga atgaagtatt tctgggatac acttttggat gctgatttgg 3121 aatgtgacat ccttggctgg cagtatatct ctgggagtat ccccgatggc cacgagcttg 3181 atcgcttgga caatcccgcg ttacaaggcg ccaaatatga cccagaaggt gagtacataa 3241 ggcaatggct tcccgagctt gcgagattgc caactgaatg gatccatcat ccatgggacg 3301 ctcctttaac cgtactcaaa gcttctggtg tggaactcgg aacaaactat gcgaaaccca 3361 ttgtagacat cgacacagct cgtgagctac tagctaaagc tatttcaaga acccgtgaag 3421 cacagatcat gatcggagca gcacctgatg agattgtagc agatagcttc gaggccttag 3481 gggctaatac cattaaagaa cctggtcttt gcccatctgt gtcttctaat gaccaacaag 3541 taccttcggc tgttcgttac aacgggtcaa agagagtgaa acctgaggaa gaagaagaga 3601 gagacatgaa gaaatctagg ggattcgatg aaagggagtt gttttcgact gctgaatctt 3661 cttcttcttc gagtgtgttt ttcgtttcgc agtcttgctc gttggcatca gaagggaaga 3721 atctggaagg tattcaagat tcatctgatc agattactac aagtttggga aaaaatggtt 3781 gcaaaggtgg cggtggctct ggaggtggtg ggtccggagg aggcggccgc atgaagctac 3841 tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag tgctccaaag 3901 aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac tctcccaaaa 3961 ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg ctagaaagac 4021 tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt ttgaaaatgg 4081 attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat aatgtgaata 4141 aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta acattgagac 4201 agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt caaagacagt 4261 tgactgtatc gggatccgcc cccccgaccg atgtcagcct gggggacgag ctccacttag 4321 acggcgagga cgtggcgatg gcgcatgccg acgcgctaga cgatttcgat ctggacatgt 4381 tgggggacgg ggattccccg ggatccaccg gatctagata actgatcata atcagccata 4441 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 4501 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 4561 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 4621 gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg 4681 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 4741 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 4801 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 4861 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 4921 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 4981 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 5041 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 5101 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 5161 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 5221 caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc 5281 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 5341 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 5401 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 5461 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 5521 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt 5581 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg 5641 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 5701 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 5761 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa 5821 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 5881 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 5941 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 6001 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 6061 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 6121 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc 6181 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 6241 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 6301 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 6361 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 6421 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 6481 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 6541 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 6601 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta 6661 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg 6721 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat 6781 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc 6841 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt 6901 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 6961 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 7021 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 7081 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 7141 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 7201 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 7261 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 7321 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 7381 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 7441 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 7501 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 7561 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 7621 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 7681 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc 7741 at //