LOCUS Exported 6830 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses CRY2 (Full length, mutated NLS) fusion with tTA2 'tet-OFF' transcriptional activator. ACCESSION . VERSION . KEYWORDS CRY-tTA (B702) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6830) 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 6830) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6830 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 11..599 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 500..519 /label=pBR322ori-F /note="pBR322 origin, forward primer" enhancer 774..1077 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1078..1281 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1232..1252 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 1338..3173 /codon_start=1 /product="Arabidopsis thaliana cryptochrome 2 in which the nuclear localization signal was removed by mutating residues 541 and 542 to alanine" /label=CRY2(Delta-NLS) /note="binds to CIB1 upon exposure to blue light (Kennedy et al., 2010)" /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKE PGLCPSVSSNDQQVPSAVRYNGSAAVKPEEEEERDMKKSRGFDERELFSTAESSSSSSV FFVSQSCSLASEGKNLEGIQDSSDQITTSLGKNGCK" CDS 3189..3935 /codon_start=1 /product="improved tetracycline-controlled transactivator" /label=tTA-Advanced /note="In the Tet-Off(R) system, tTA-Advanced binds to the Tet-responsive element and stimulates transcription only in the absence of tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" polyA_signal 4057..4178 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 4331..4452 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" rep_origin complement(4459..4914) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4596..4617) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4808..4827 /label=F1ori-R /note="F1 origin, reverse primer" promoter 4941..5045 /gene="bla" /label=AmpR promoter promoter 5047..5404 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(5070..5090) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 5255..5390 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5317..5336 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5439..6233 /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(5493..5512) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6103..6122 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(6421..6440) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 6465..6512 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" ORIGIN 1 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 61 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 121 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 181 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 241 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 301 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 361 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 421 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 481 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 541 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 601 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 661 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat 721 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca 781 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca 841 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg 901 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg 961 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc 1021 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg 1081 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca 1141 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt 1201 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg 1261 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact 1321 cagatctcga ggccaccatg aagatggaca aaaagactat agtttggttt agaagagacc 1381 taaggattga ggataatcct gcattagcag cagctgctca cgaaggatct gtttttcctg 1441 tcttcatttg gtgtcctgaa gaagaaggac agttttatcc tggaagagct tcaagatggt 1501 ggatgaaaca atcacttgct cacttatctc aatccttgaa ggctcttgga tctgacctca 1561 ctttaatcaa aacccacaac acgatttcag cgatcttgga ttgtatccgc gttaccggtg 1621 ctacaaaagt cgtctttaac cacctctatg atcctgtttc gttagttcgg gaccataccg 1681 taaaggagaa gctggtggaa cgtgggatct ctgtgcaaag ctacaatgga gatctattgt 1741 atgaaccgtg ggagatatac tgcgaaaagg gcaaaccttt tacgagtttc aattcttact 1801 ggaagaaatg cttagatatg tcgattgaat ccgttatgct tcctcctcct tggcggttga 1861 tgccaataac tgcagcggct gaagcgattt gggcgtgttc gattgaagaa ctagggctgg 1921 agaatgaggc cgagaaaccg agcaatgcgt tgttaactag agcttggtct ccaggatgga 1981 gcaatgctga taagttacta aatgagttca tcgagaagca gttgatagat tatgcaaaga 2041 acagcaagaa agttgttggg aattctactt cactactttc tccgtatctc catttcgggg 2101 aaataagcgt cagacacgtt ttccagtgtg cccggatgaa acaaattata tgggcaagag 2161 ataagaacag tgaaggagaa gaaagtgcag atctttttct taggggaatc ggtttaagag 2221 agtattctcg gtatatatgt ttcaacttcc cgtttactca cgagcaatcg ttgttgagtc 2281 atcttcggtt tttcccttgg gatgctgatg ttgataagtt caaggcctgg agacaaggca 2341 ggaccggtta tccgttggtg gatgccggaa tgagagagct ttgggctacc ggatggatgc 2401 ataacagaat aagagtgatt gtttcaagct ttgctgtgaa gtttcttctc cttccatgga 2461 aatggggaat gaagtatttc tgggatacac ttttggatgc tgatttggaa tgtgacatcc 2521 ttggctggca gtatatctct gggagtatcc ccgatggcca cgagcttgat cgcttggaca 2581 atcccgcgtt acaaggcgcc aaatatgacc cagaaggtga gtacataagg caatggcttc 2641 ccgagcttgc gagattgcca actgaatgga tccatcatcc atgggacgct cctttaaccg 2701 tactcaaagc ttctggtgtg gaactcggaa caaactatgc gaaacccatt gtagacatcg 2761 acacagctcg tgagctacta gctaaagcta tttcaagaac ccgtgaagca cagatcatga 2821 tcggagcagc acctgatgag attgtagcag atagcttcga ggccttaggg gctaatacca 2881 ttaaagaacc tggtctttgc ccatctgtgt cttctaatga ccaacaagta ccttcggctg 2941 ttcgttacaa cgggtcagcg gcagtgaaac ctgaggaaga agaagagaga gacatgaaga 3001 aatctagggg attcgatgaa agggagttgt tttcgactgc tgaatcttct tcttcttcga 3061 gtgtgttttt cgtttcgcag tcttgctcgt tggcatcaga agggaagaat ctggaaggta 3121 ttcaagattc atctgatcag attactacaa gtttgggaaa aaatggttgc aaagcccggg 3181 ccgccaccat gtctagactg gacaagagca aagtcataaa ctctgctctg gaattactca 3241 atgaagtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc 3301 agcctaccct gtactggcac gtgaagaaca agcgggccct gctcgatgcc ctggcaatcg 3361 agatgctgga caggcatcat acccacttct gccccctgga aggcgagtca tggcaagact 3421 ttctgcggaa caacgccaag tcattccgct gtgctctcct ctcacatcgc gacggggcta 3481 aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa aatcagctcg 3541 cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg tccgccgtgg 3601 gccactttac actgggctgc gtattggagg atcaggagca tcaagtagca aaagaggaaa 3661 gagagacacc taccaccgat tctatgcccc cacttctgag acaagcaatt gagctgttcg 3721 accatcaggg agccgaacct gccttccttt tcggcctgga actaatcata tgtggcctgg 3781 agaaacagct aaagtgcgaa agcggcgggc cggccgacgc ccttgacgat tttgacttag 3841 acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc 3901 ttgacgattt tgaccttgac atgctccccg gatgagaatt ctagataact gatcataatc 3961 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg 4021 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat 4081 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 4141 tctagttgtg gtttgtccaa actcatcaat gtatcttatt taaattgggc gcgcccaact 4201 agttctagag cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt 4261 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat 4321 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 4381 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 4441 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt 4501 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat 4561 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat 4621 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac 4681 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc 4741 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga 4801 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 4861 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg 4921 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 4981 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 5041 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc 5101 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag 5161 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 5221 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 5281 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 5341 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 5401 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc 5461 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga 5521 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt 5581 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat 5641 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg 5701 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg 5761 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc 5821 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga 5881 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag 5941 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc 6001 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg 6061 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata 6121 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg 6181 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac 6241 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc 6301 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat 6361 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac 6421 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca 6481 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg 6541 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt 6601 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc 6661 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca 6721 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 6781 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca //