LOCUS       sequence-176938-        6487 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses CRY2 (Full length, mutant NLS) fused to Gal4DNA (residues 
            1-147) fused to VP16 activation domain (long form).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6487)
  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 6487)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2017 Apr 20. doi: 10.1093/nar/gkx260."
COMMENT     Sequence Label: CRY-GalVP16l (B694)
FEATURES             Location/Qualifiers
     source          1..6487
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     CDS             join(625..2244,2245..2250,2251..2460)
                     /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             2470..2910
                     /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"
     CDS             2917..3150
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes 
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     polyA_signal    3275..3396
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3312..3331)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3366..3385
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3403..3858)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3540..3561)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3752..3771
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3885..3989
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3991..4348
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3991..4182
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4014..4034)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4199..4334
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4383..5177
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4383..5177
                     /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(4437..4456)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5047..5066
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5365..5384)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5409..5456
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5785..6373
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6274..6293
                     /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 ccggactcag atctcgaggc caccatgaag atggacaaaa agactatagt ttggtttaga
      661 agagacctaa ggattgagga taatcctgca ttagcagcag ctgctcacga aggatctgtt
      721 tttcctgtct tcatttggtg tcctgaagaa gaaggacagt tttatcctgg aagagcttca
      781 agatggtgga tgaaacaatc acttgctcac ttatctcaat ccttgaaggc tcttggatct
      841 gacctcactt taatcaaaac ccacaacacg atttcagcga tcttggattg tatccgcgtt
      901 accggtgcta caaaagtcgt ctttaaccac ctctatgatc ctgtttcgtt agttcgggac
      961 cataccgtaa aggagaagct ggtggaacgt gggatctctg tgcaaagcta caatggagat
     1021 ctattgtatg aaccgtggga gatatactgc gaaaagggca aaccttttac gagtttcaat
     1081 tcttactgga agaaatgctt agatatgtcg attgaatccg ttatgcttcc tcctccttgg
     1141 cggttgatgc caataactgc agcggctgaa gcgatttggg cgtgttcgat tgaagaacta
     1201 gggctggaga atgaggccga gaaaccgagc aatgcgttgt taactagagc ttggtctcca
     1261 ggatggagca atgctgataa gttactaaat gagttcatcg agaagcagtt gatagattat
     1321 gcaaagaaca gcaagaaagt tgttgggaat tctacttcac tactttctcc gtatctccat
     1381 ttcggggaaa taagcgtcag acacgttttc cagtgtgccc ggatgaaaca aattatatgg
     1441 gcaagagata agaacagtga aggagaagaa agtgcagatc tttttcttag gggaatcggt
     1501 ttaagagagt attctcggta tatatgtttc aacttcccgt ttactcacga gcaatcgttg
     1561 ttgagtcatc ttcggttttt cccttgggat gctgatgttg ataagttcaa ggcctggaga
     1621 caaggcagga ccggttatcc gttggtggat gccggaatga gagagctttg ggctaccgga
     1681 tggatgcata acagaataag agtgattgtt tcaagctttg ctgtgaagtt tcttctcctt
     1741 ccatggaaat ggggaatgaa gtatttctgg gatacacttt tggatgctga tttggaatgt
     1801 gacatccttg gctggcagta tatctctggg agtatccccg atggccacga gcttgatcgc
     1861 ttggacaatc ccgcgttaca aggcgccaaa tatgacccag aaggtgagta cataaggcaa
     1921 tggcttcccg agcttgcgag attgccaact gaatggatcc atcatccatg ggacgctcct
     1981 ttaaccgtac tcaaagcttc tggtgtggaa ctcggaacaa actatgcgaa acccattgta
     2041 gacatcgaca cagctcgtga gctactagct aaagctattt caagaacccg tgaagcacag
     2101 atcatgatcg gagcagcacc tgatgagatt gtagcagata gcttcgaggc cttaggggct
     2161 aataccatta aagaacctgg tctttgccca tctgtgtctt ctaatgacca acaagtacct
     2221 tcggctgttc gttacaacgg gtcagcggca gtgaaacctg aggaagaaga agagagagac
     2281 atgaagaaat ctaggggatt cgatgaaagg gagttgtttt cgactgctga atcttcttct
     2341 tcttcgagtg tgtttttcgt ttcgcagtct tgctcgttgg catcagaagg gaagaatctg
     2401 gaaggtattc aagattcatc tgatcagatt actacaagtt tgggaaaaaa tggttgcaaa
     2461 gcccgggaca tgaagctact gtcttctatc gaacaagcat gcgatatttg ccgacttaaa
     2521 aagctcaagt gctccaaaga aaaaccgaag tgcgccaagt gtctgaagaa caactgggag
     2581 tgtcgctact ctcccaaaac caaaaggtct ccgctgacta gggcacatct gacagaagtg
     2641 gaatcaaggc tagaaagact ggaacagcta tttctactga tttttcctcg agaagacctt
     2701 gacatgattt tgaaaatgga ttctttacag gatataaaag cattgttaac aggattattt
     2761 gtacaagata atgtgaataa agatgccgtc acagatagat tggcttcagt ggagactgat
     2821 atgcctctaa cattgagaca gcatagaata agtgcgacat catcatcgga agagagtagt
     2881 aacaaaggtc aaagacagtt gactgtatcg ggatccgccc ccccgaccga tgtcagcctg
     2941 ggggacgagc tccacttaga cggcgaggac gtggcgatgg cgcatgccga cgcgctagac
     3001 gatttcgatc tggacatgtt gggggacggg gattccccgg gtccgggatt taccccccac
     3061 gactccgccc cctacggcgc tctggatatg gccgacttcg agtttgagca gatgtttacc
     3121 gatgcccttg gaattgacga gtacggtggg tgagcggccg cgactctaga tcataatcag
     3181 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     3241 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     3301 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3361 tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata
     3421 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     3481 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     3541 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     3601 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     3661 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     3721 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     3781 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     3841 cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3901 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3961 tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt
     4021 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4081 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     4141 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     4201 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4261 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4321 cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc
     4381 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     4441 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     4501 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     4561 tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     4621 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     4681 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     4741 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     4801 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     4861 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
     4921 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     4981 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     5041 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     5101 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     5161 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     5221 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5281 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5341 cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     5401 cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa
     5461 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
     5521 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
     5581 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat
     5641 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5701 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5761 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5821 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5881 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     5941 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6001 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6061 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6121 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6181 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6241 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6301 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6361 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6421 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6481 catgcat
//
