LOCUS       sequence-59274-e        6826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses optogenetically controlled RhoA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6826)
  AUTHORS   Bugaj LJ, Choksi AT, Mesuda CK, Kane RS, Schaffer DV
  TITLE     Optogenetic protein clustering and signaling activation in mammalian
            cells.
  JOURNAL   Nat Methods. 2013 Feb 3. doi: 10.1038/nmeth.2360.
  PUBMED    23377377
REFERENCE   2  (bases 1 to 6826)
  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: "Nat 
            Methods. 2013 Feb 3. doi: 10.1038/nmeth.2360."
COMMENT     Sequence Label: pCry2PHR-mCh-RhoA
FEATURES             Location/Qualifiers
     source          1..6826
                     /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             625..2118
                     /codon_start=1
                     /product="N-terminal photolyase homology region of 
                     Arabidopsis thaliana cryptochrome 2"
                     /label=CRY2PHR
                     /note="binds to CIB1 upon exposure to blue light (Kennedy 
                     et al., 2010)"
                     /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
                     FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
                     DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
                     ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
                     EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE
                     ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
                     VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
                     ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
                     SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAA"
     regulatory      2137..2146
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2143..2850
                     /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(2288..2306)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2556..2575
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2881..3459
                     /codon_start=1
                     /product="human Rho family small GTPase RhoA"
                     /label=RhoA
                     /translation="AAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADI
                     EVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKH
                     FCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTK
                     DGVREVFEMATRAALQARRGKKKSGCLVL"
     polyA_signal    3614..3735
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3651..3670)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3705..3724
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3742..4197)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3879..3900)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4091..4110
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4224..4328
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4330..4687
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4330..4521
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4353..4373)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4538..4673
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4722..5516
                     /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             4722..5516
                     /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(4776..4795)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5386..5405
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5704..5723)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5748..5795
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6124..6712
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6613..6632
                     /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 gagcagcagc ccgggatcca ccggtcgcca ccatggtgag caagggcgag
     2161 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     2221 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     2281 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     2341 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     2401 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     2461 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     2521 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     2581 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     2641 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     2701 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     2761 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     2821 tccaccggcg gcatggacga gctgtacaag ggtggaggag gtagtggagg aggaggatca
     2881 gctgccatcc ggaagaaact ggtgattgtt ggtgatggag cctgtggaaa gacatgcttg
     2941 ctcatagtct tcagcaagga ccagttccca gaggtgtatg tgcccacagt gtttgagaac
     3001 tatgtggcag atatcgaggt ggatggaaag caggtagagt tggctttgtg ggacacagct
     3061 gggcaggaag attatgaccg cctgaggccc ctctcctacc cagataccga tgttatactg
     3121 atgtgttttt ccatcgacag ccctgatagt ttagaaaaca tcccagaaaa gtggacccca
     3181 gaagtcaagc atttctgtcc caacgtgccc atcatcctgg ttgggaataa gaaggatctt
     3241 cggaatgatg agcacacaag gcgggagcta gccaagatga agcaggagcc ggtgaaacct
     3301 gaagaaggca gagatatggc aaacaggatt ggcgcttttg ggtacatgga gtgttcagca
     3361 aagaccaaag atggagtgag agaggttttt gaaatggcta cgagagctgc tctgcaagct
     3421 agacgtggga agaaaaaatc tgggtgcctt gtcttgtgaa accttgctgg aagcacagcc
     3481 cttatgcggt aagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt
     3541 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     3601 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3661 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3721 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     3781 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     3841 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     3901 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     3961 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     4021 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     4081 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     4141 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     4201 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4261 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4321 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     4381 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4441 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4501 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4561 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4621 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4681 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     4741 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4801 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4861 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
     4921 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4981 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     5041 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     5101 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     5161 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     5221 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
     5281 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     5341 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     5401 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     5461 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     5521 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     5581 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     5641 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     5701 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     5761 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     5821 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     5881 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     5941 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
     6001 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6061 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6121 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6181 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6241 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     6301 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6361 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6421 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6481 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6541 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6601 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6661 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6721 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     6781 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
