LOCUS       Exported                7373 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses CRY2 (full-length, with endogenous NLS) fused to Gal4 
            (aa1-65 of Gal4BD), downstream of mCherry-IRES.
ACCESSION   .
VERSION     .
KEYWORDS    CRY-Gal∆DD (B1013)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7373)
  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 7373)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7373
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        60..363
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        364..567
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     518..538
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      606..615
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             612..1319
                     /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(757..775)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1025..1044
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1921..1937)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1922..1938)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     CDS             1949..3784
                     /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"
     polyA_signal    4160..4281
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4197..4216)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4251..4270
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4288..4743)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4425..4446)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4637..4656
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4770..4874
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4876..5233
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(4899..4919)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5084..5219
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5146..5165
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5268..6062
                     /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(5322..5341)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5932..5951
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6250..6269)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6294..6341
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6670..7258
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7159..7178
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
       61 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      121 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      181 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      241 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      301 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      361 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      421 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      481 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      541 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg ctagcgctac
      601 cggtcgccac catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
      661 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
      721 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
      781 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
      841 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
      901 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
      961 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     1021 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     1081 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     1141 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     1201 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     1261 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     1321 gtggcggcta gtactccggt attgcggtac ccttgtacgc ctgttttata ctcccttccc
     1381 gtaacttaga cgcacaaaac caagttcaat agaagggggt acaaaccagt accaccacga
     1441 acaagcactt ctgtttcccc ggtgatgtcg tatagactgc ttgcgtggtt gaaagcgacg
     1501 gatccgttat ccgcttatgt acttcgagaa gcccagtacc acctcggaat cttcgatgcg
     1561 ttgcgctcag cactcaaccc cagagtgtag cttaggctga tgagtctgga catccctcac
     1621 cggtgacggt ggtccaggct gcgttggcgg cctacctatg gctaacgcca tgggacgcta
     1681 gttgtgaaca aggtgtgaag agcctattga gctacataag aatcctccgg cccctgaatg
     1741 cggctaatcc caacctcgga gcaggtggtc acaaaccagt gattggcctg tcgtaacgcg
     1801 caagtccgtg gcggaaccga ctactttggg tgtccgtgtt tccttttatt ttattgtggc
     1861 tgcttatggt gacaatcaca gattgttatc ataaagcgaa ttggatagga tcaagcttat
     1921 cgataccgtc gacctcgagc tcgccaccat gaagatggac aaaaagacta tagtttggtt
     1981 tagaagagac ctaaggattg aggataatcc tgcattagca gcagctgctc acgaaggatc
     2041 tgtttttcct gtcttcattt ggtgtcctga agaagaagga cagttttatc ctggaagagc
     2101 ttcaagatgg tggatgaaac aatcacttgc tcacttatct caatccttga aggctcttgg
     2161 atctgacctc actttaatca aaacccacaa cacgatttca gcgatcttgg attgtatccg
     2221 cgttaccggt gctacaaaag tcgtctttaa ccacctctat gatcctgttt cgttagttcg
     2281 ggaccatacc gtaaaggaga agctggtgga acgtgggatc tctgtgcaaa gctacaatgg
     2341 agatctattg tatgaaccgt gggagatata ctgcgaaaag ggcaaacctt ttacgagttt
     2401 caattcttac tggaagaaat gcttagatat gtcgattgaa tccgttatgc ttcctcctcc
     2461 ttggcggttg atgccaataa ctgcagcggc tgaagcgatt tgggcgtgtt cgattgaaga
     2521 actagggctg gagaatgagg ccgagaaacc gagcaatgcg ttgttaacta gagcttggtc
     2581 tccaggatgg agcaatgctg ataagttact aaatgagttc atcgagaagc agttgataga
     2641 ttatgcaaag aacagcaaga aagttgttgg gaattctact tcactacttt ctccgtatct
     2701 ccatttcggg gaaataagcg tcagacacgt tttccagtgt gcccggatga aacaaattat
     2761 atgggcaaga gataagaaca gtgaaggaga agaaagtgca gatctttttc ttaggggaat
     2821 cggtttaaga gagtattctc ggtatatatg tttcaacttc ccgtttactc acgagcaatc
     2881 gttgttgagt catcttcggt ttttcccttg ggatgctgat gttgataagt tcaaggcctg
     2941 gagacaaggc aggaccggtt atccgttggt ggatgccgga atgagagagc tttgggctac
     3001 cggatggatg cataacagaa taagagtgat tgtttcaagc tttgctgtga agtttcttct
     3061 ccttccatgg aaatggggaa tgaagtattt ctgggataca cttttggatg ctgatttgga
     3121 atgtgacatc cttggctggc agtatatctc tgggagtatc cccgatggcc acgagcttga
     3181 tcgcttggac aatcccgcgt tacaaggcgc caaatatgac ccagaaggtg agtacataag
     3241 gcaatggctt cccgagcttg cgagattgcc aactgaatgg atccatcatc catgggacgc
     3301 tcctttaacc gtactcaaag cttctggtgt ggaactcgga acaaactatg cgaaacccat
     3361 tgtagacatc gacacagctc gtgagctact agctaaagct atttcaagaa cccgtgaagc
     3421 acagatcatg atcggagcag cacctgatga gattgtagca gatagcttcg aggccttagg
     3481 ggctaatacc attaaagaac ctggtctttg cccatctgtg tcttctaatg accaacaagt
     3541 accttcggct gttcgttaca acgggtcaaa gagagtgaaa cctgaggaag aagaagagag
     3601 agacatgaag aaatctaggg gattcgatga aagggagttg ttttcgactg ctgaatcttc
     3661 ttcttcttcg agtgtgtttt tcgtttcgca gtcttgctcg ttggcatcag aagggaagaa
     3721 tctggaaggt attcaagatt catctgatca gattactaca agtttgggaa aaaatggttg
     3781 caaaggtggc ggtggctctg gaggtggtgg gtccggagga ggcggccgca tgaagctact
     3841 gtcttctatc gaacaagcat gcgatatttg ccgacttaaa aagctcaagt gctccaaaga
     3901 aaaaccgaag tgcgccaagt gtctgaagaa caactgggag tgtcgctact ctcccaaaac
     3961 caaaaggtct ccgctgacta gggcacatct gacagaagtg gaatcaaggc tagaaagact
     4021 ggaatctagg ggatccaccg gatctagata actgatcata atcagccata ccacatttgt
     4081 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     4141 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     4201 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4261 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     4321 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     4381 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     4441 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     4501 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     4561 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     4621 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     4681 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     4741 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4801 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4861 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     4921 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4981 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     5041 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     5101 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     5161 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     5221 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     5281 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     5341 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     5401 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     5461 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5521 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5581 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5641 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5701 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5761 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     5821 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     5881 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     5941 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     6001 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     6061 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     6121 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     6181 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     6241 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     6301 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     6361 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     6421 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     6481 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     6541 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6601 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6661 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6721 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6781 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6841 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6901 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6961 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7021 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7081 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7141 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7201 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7261 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7321 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc att
//
