LOCUS       sequence-183889-        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"
     rep_origin      16..604
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     505..524
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        779..1082
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1083..1286
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1237..1257
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1343..2962,2963..2968,2969..3178)
                     /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             3188..3628
                     /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             3635..3868
                     /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    3993..4114
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4030..4049)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4084..4103
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4121..4576)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4258..4279)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4470..4489
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4603..4707
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4709..5066
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4709..4900
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4732..4752)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4917..5052
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5101..5895
                     /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             5101..5895
                     /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(5155..5174)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5765..5784
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6083..6102)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6127..6174
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
       61 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
      121 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
      181 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
      241 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
      301 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
      361 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
      421 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
      481 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
      541 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
      601 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
      661 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta
      721 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      781 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      841 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      901 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      961 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     1021 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     1081 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     1141 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     1201 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     1261 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc
     1321 ggactcagat ctcgaggcca ccatgaagat ggacaaaaag actatagttt ggtttagaag
     1381 agacctaagg attgaggata atcctgcatt agcagcagct gctcacgaag gatctgtttt
     1441 tcctgtcttc atttggtgtc ctgaagaaga aggacagttt tatcctggaa gagcttcaag
     1501 atggtggatg aaacaatcac ttgctcactt atctcaatcc ttgaaggctc ttggatctga
     1561 cctcacttta atcaaaaccc acaacacgat ttcagcgatc ttggattgta tccgcgttac
     1621 cggtgctaca aaagtcgtct ttaaccacct ctatgatcct gtttcgttag ttcgggacca
     1681 taccgtaaag gagaagctgg tggaacgtgg gatctctgtg caaagctaca atggagatct
     1741 attgtatgaa ccgtgggaga tatactgcga aaagggcaaa ccttttacga gtttcaattc
     1801 ttactggaag aaatgcttag atatgtcgat tgaatccgtt atgcttcctc ctccttggcg
     1861 gttgatgcca ataactgcag cggctgaagc gatttgggcg tgttcgattg aagaactagg
     1921 gctggagaat gaggccgaga aaccgagcaa tgcgttgtta actagagctt ggtctccagg
     1981 atggagcaat gctgataagt tactaaatga gttcatcgag aagcagttga tagattatgc
     2041 aaagaacagc aagaaagttg ttgggaattc tacttcacta ctttctccgt atctccattt
     2101 cggggaaata agcgtcagac acgttttcca gtgtgcccgg atgaaacaaa ttatatgggc
     2161 aagagataag aacagtgaag gagaagaaag tgcagatctt tttcttaggg gaatcggttt
     2221 aagagagtat tctcggtata tatgtttcaa cttcccgttt actcacgagc aatcgttgtt
     2281 gagtcatctt cggtttttcc cttgggatgc tgatgttgat aagttcaagg cctggagaca
     2341 aggcaggacc ggttatccgt tggtggatgc cggaatgaga gagctttggg ctaccggatg
     2401 gatgcataac agaataagag tgattgtttc aagctttgct gtgaagtttc ttctccttcc
     2461 atggaaatgg ggaatgaagt atttctggga tacacttttg gatgctgatt tggaatgtga
     2521 catccttggc tggcagtata tctctgggag tatccccgat ggccacgagc ttgatcgctt
     2581 ggacaatccc gcgttacaag gcgccaaata tgacccagaa ggtgagtaca taaggcaatg
     2641 gcttcccgag cttgcgagat tgccaactga atggatccat catccatggg acgctccttt
     2701 aaccgtactc aaagcttctg gtgtggaact cggaacaaac tatgcgaaac ccattgtaga
     2761 catcgacaca gctcgtgagc tactagctaa agctatttca agaacccgtg aagcacagat
     2821 catgatcgga gcagcacctg atgagattgt agcagatagc ttcgaggcct taggggctaa
     2881 taccattaaa gaacctggtc tttgcccatc tgtgtcttct aatgaccaac aagtaccttc
     2941 ggctgttcgt tacaacgggt cagcggcagt gaaacctgag gaagaagaag agagagacat
     3001 gaagaaatct aggggattcg atgaaaggga gttgttttcg actgctgaat cttcttcttc
     3061 ttcgagtgtg tttttcgttt cgcagtcttg ctcgttggca tcagaaggga agaatctgga
     3121 aggtattcaa gattcatctg atcagattac tacaagtttg ggaaaaaatg gttgcaaagc
     3181 ccgggacatg aagctactgt cttctatcga acaagcatgc gatatttgcc gacttaaaaa
     3241 gctcaagtgc tccaaagaaa aaccgaagtg cgccaagtgt ctgaagaaca actgggagtg
     3301 tcgctactct cccaaaacca aaaggtctcc gctgactagg gcacatctga cagaagtgga
     3361 atcaaggcta gaaagactgg aacagctatt tctactgatt tttcctcgag aagaccttga
     3421 catgattttg aaaatggatt ctttacagga tataaaagca ttgttaacag gattatttgt
     3481 acaagataat gtgaataaag atgccgtcac agatagattg gcttcagtgg agactgatat
     3541 gcctctaaca ttgagacagc atagaataag tgcgacatca tcatcggaag agagtagtaa
     3601 caaaggtcaa agacagttga ctgtatcggg atccgccccc ccgaccgatg tcagcctggg
     3661 ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg cgctagacga
     3721 tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta ccccccacga
     3781 ctccgccccc tacggcgctc tggatatggc cgacttcgag tttgagcaga tgtttaccga
     3841 tgcccttgga attgacgagt acggtgggtg agcggccgcg actctagatc ataatcagcc
     3901 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     3961 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     4021 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4081 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     4141 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4201 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     4261 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4321 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     4381 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4441 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4501 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4561 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4621 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4681 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     4741 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4801 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4861 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4921 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4981 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     5041 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     5101 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     5161 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     5221 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     5281 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     5341 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     5401 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     5461 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     5521 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     5581 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     5641 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     5701 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     5761 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     5821 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     5881 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     5941 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     6001 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     6061 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     6121 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     6181 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     6241 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     6301 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     6361 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6421 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6481 tagaaaa
//
