LOCUS       sequence-341721-        7417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mCherry-NES-SspB-Tiam-DHPH-P2A-iLIDcaax 
            (opto-Rac1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7417)
  AUTHORS   Inaba H, Miao Q, Nakata T
  TITLE     Optogenetic control of small GTPases reveals RhoA mediates 
            intracellular calcium signaling.
  JOURNAL   J Biol Chem. 2021 Jan-Jun;296:100290. doi: 
            10.1016/j.jbc.2021.100290. Epub 2021 Jan 13.
  PUBMED    33453281
REFERENCE   2  (bases 1 to 7417)
  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: "J Biol 
            Chem. 2021 Jan-Jun;296:100290. doi: 10.1016/j.jbc.2021.100290. Epub 
            2021 Jan 13."
COMMENT     Sequence Label: mCherry-NES-SspB-Tiam-DHPH-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..7417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        122..425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        426..629
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     580..600
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      668..677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             674..1381
                     /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(819..837)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1087..1106
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1430..1759
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein"
                     /label=SspB nano
                     /note="dimerizes with iLID at high affinity to create iLID 
                     nano (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNARFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     CDS             3566..3622
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3644..4081
                     /codon_start=1
                     /product="improved light-inducible dimer when used with 
                     SspB nano or SspB micro (Guntas et al., 2015)"
                     /label=iLID
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNVFHLQPMRDYKGDVQ
                     YFIGVQLDGTERLHGAAEREAVCLIKKTAFQIAEAANDENYF"
     polyA_signal    4266..4387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4303..4322)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4357..4376
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4394..4849)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4531..4552)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4743..4762
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4876..4980
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4982..5339
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4982..5173
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5005..5025)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5190..5325
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5374..6168
                     /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             5374..6168
                     /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(5428..5447)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6038..6057
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6356..6375)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6400..6447
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6776..7364
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7265..7284
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
       61 ttagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      121 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      181 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      241 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      301 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      361 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      421 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      481 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      541 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      601 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct
      661 accggtcgcc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
      721 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
      781 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
      841 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
      901 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
      961 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     1021 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     1081 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     1141 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     1201 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     1261 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     1321 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     1381 gttcggcatt gatctgagcg gcctgaccct gggtaccagt gctggaggta gctccccgaa
     1441 acgccctaag ctgctgcgtg aatattacga ttggctggtt gataacagct ttaccccata
     1501 tctggtggtg gatgccacat acctgggcgt gaacgtgccc gtggagtatg tgaaagacgg
     1561 tcagatcgtg ctgaatctgt ctgcaagtgc gaccggcaac ctgcaactga caaatgattt
     1621 tatccagttc aacgcccgct ttaagggcgt gtctcgtgaa ctgtatatcc cgatgggtgc
     1681 cgctctggcc atttacgctc gcgagaacgg cgatggtgtg atgttcgaac cagaagaaat
     1741 ctatgacgag ctgaatattg gtctcgacag tgctggaggt agtgctggag gttcagctgg
     1801 tggactcgag atgaacccct ctgaccagag cccatctcct caggactcca cggggcctca
     1861 gctggcgacc atgagacaac tctcggatgc agataagctg cgcaaggtga tctgcgagct
     1921 cctggagacg gagcgcacct acgtgaagga tttaaactgt cttatggaga gatacctaaa
     1981 gcctcttcaa aaagaaactt ttctcaccca ggatgagctt gacgtgcttt ttggaaattt
     2041 aacggaaatg gtagagtttc aagtagaatt ccttaaaact ctagaagatg gagtgagact
     2101 ggtacctgat ttggaaaagc ttgagaaggt tgatcaattt aagaaagtgc tgttctctct
     2161 ggggggatca ttcctgtatt atgctgaccg cttcaagctc tacagtgcct tctgcgccag
     2221 ccacacaaaa gttcccaagg tcctggtgaa agccaagaca gacacggctt tcaaggcatt
     2281 cttggatgcc cagaacccga agcagcagca ctcatccacg ctggagtcgt acctcatcaa
     2341 gcccatccag aggatcctca agtacccact tctgctcagg gagctgttcg ccctgaccga
     2401 tgcggagagc gaggagcact accacctgga cgtggccatc aagaccatga acaaggttgc
     2461 cagtcacatc aatgagatgc agaaaatcca tgaagagttt ggggctgtgt ttgaccagct
     2521 gattgctgaa cagactggtg ggaaaaaaga ggttgcagat ctgagcatgg gagacctgct
     2581 tttgcacact accgtgatct ggctgaaccc gccggcctcg ctgggcaagt ggaaaaagga
     2641 accagagttg gcagcattcg tcttcaaaac tgctgtggtc cttgtgtata aagatggttc
     2701 caaacagaag aagaaacttg taggatctca caggctttcc atttatgagg actgggaccc
     2761 cttcagattt cgacacatga tccccacgga agcgctgcag gttcgagctt tggcgagtgc
     2821 agatgcagag gcaaatgccg tgtgtgaaat tgtccatgta aaatccgagt ctgaagggag
     2881 gccggagagg gtctttcact tgtgctgcag ctccccagag agccgaaagg atttcctaaa
     2941 ggctgtgcat tcaatcctgc gtgataagca cagaagacag ctcctcaaaa ccgagagcct
     3001 tccctcatcc cagcaatatg tcccttttgg aggcaaaaga ttgtgtgcac tgaagggggc
     3061 caggccggcc atgagcaggg cagtgtctgc cccaagcaag tctcttggga ggaggaggcg
     3121 gcggctggct cgaaacaggt ttaccattga ttctgatgcc gtctccgcaa gcagcccgga
     3181 gaaagagtcc cagcagcccc ccggtggcgg ggacactgac cgatgggtag aggagcagtt
     3241 tgatcttgct cagtatgagg agcaagatga catcaaggag acagacatcc tcagtgacga
     3301 tgatgagttc tgtgagtccg tgaagggtgc ctcagtggac agagacctgc aggagcggct
     3361 tcaggccacc tccatcagtc agcgggaaag aggccggaaa accctggata gtcacgcgtc
     3421 ccgcatggca cagctcaaga agcaagctgc cctgtcgggg atcaatggag gcctggagag
     3481 cgcaagcgag gaagtcattt gggttaggcg tgaagacttt gccccctcca ggaaactgaa
     3541 cactgagatc ccgcggggaa gcggagctac taacttcagc ctgctgaagc aggctggaga
     3601 cgtggaggag aaccctggac ctgcaccggg atccggggag tttctggcaa ccacactgga
     3661 acggatcgag aaaaatttcg tgattactga tccgagactg cctgacaacc caatcatttt
     3721 tgcgagcgat tccttcctgc agctgacaga atattctcgg gaagagatcc tggggcgcaa
     3781 ttgccgtttt ctgcagggac ccgagacaga ccgtgccact gttcggaaaa tcagagatgc
     3841 tattgacaac cagactgaag tgaccgttca gctgatcaat tataccaaga gcggcaagaa
     3901 gttctggaac gtgttccacc tgcagccgat gcgcgattat aagggcgacg tccagtactt
     3961 cattggcgtg cagctggatg gcaccgaacg tcttcatggc gccgctgagc gtgaggcggt
     4021 ctgcctgatc aaaaagacag cctttcagat tgctgaggca gcgaacgacg aaaattactt
     4081 tggaagcggg agtgggagca aaaagaagaa aaagaagtca aagacaaagt gtgtaattat
     4141 gtaggcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     4201 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     4261 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4321 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4381 tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     4441 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     4501 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     4561 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     4621 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     4681 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4741 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4801 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     4861 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4921 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4981 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     5041 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     5101 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     5161 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     5221 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     5281 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     5341 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     5401 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     5461 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     5521 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     5581 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     5641 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     5701 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     5761 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     5821 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     5881 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     5941 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     6001 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     6061 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     6121 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     6181 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     6241 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     6301 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     6361 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     6421 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     6481 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     6541 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     6601 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     6661 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6721 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6781 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6841 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6901 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6961 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7021 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7081 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7141 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7201 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7261 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7321 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7381 cctttttacg gttcctggcc ttttgctggc cttttgc
//
