LOCUS       sequence-341727-        7135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mCherry-NES-SspB-SOS2cat-P2A-iLIDcaax 
            (opto-Ras).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7135)
  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 7135)
  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-SOS2cat-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..7135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        118..421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        422..625
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     576..596
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      664..673
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             670..1377
                     /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(815..833)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1083..1102
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1426..1755
                     /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             3280..3336
                     /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             3358..3795
                     /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    3980..4101
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4017..4036)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4071..4090
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4108..4563)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4245..4266)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4457..4476
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4590..4694
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4696..5053
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4696..4887
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4719..4739)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4904..5039
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5088..5882
                     /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             5088..5882
                     /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(5142..5161)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5752..5771
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6070..6089)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6114..6161
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6490..7078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6979..6998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcattag
       61 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      121 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      181 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      241 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      301 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      361 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      421 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      481 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      541 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      601 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct agcgctaccg
      661 gtcgccacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
      721 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
      781 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
      841 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
      901 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
      961 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     1021 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     1081 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     1141 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     1201 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     1261 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     1321 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagttc
     1381 ggcattgatc tgagcggcct gaccctgggt accagtgctg gaggtagctc cccgaaacgc
     1441 cctaagctgc tgcgtgaata ttacgattgg ctggttgata acagctttac cccatatctg
     1501 gtggtggatg ccacatacct gggcgtgaac gtgcccgtgg agtatgtgaa agacggtcag
     1561 atcgtgctga atctgtctgc aagtgcgacc ggcaacctgc aactgacaaa tgattttatc
     1621 cagttcaacg cccgctttaa gggcgtgtct cgtgaactgt atatcccgat gggtgccgct
     1681 ctggccattt acgctcgcga gaacggcgat ggtgtgatgt tcgaaccaga agaaatctat
     1741 gacgagctga atattggtct cgacagtgct ggaggtagtg ctggaggttc agctggtgga
     1801 ctcgagcaac cactgagatt accaagtcct gaagtatatc gttttgtagt aaaagactct
     1861 gaggaaaaca ttgtttttga agacaacttg caaagtagaa gtggcatccc cattattaaa
     1921 ggaggaactg tagtgaaatt aattgaaagg ttaacatatc atatgtatgc agatcccaat
     1981 tttgttcgta cttttcttac cacatatcgt tcattttgta aaccacagga attgctgagc
     2041 ttactgattg aacggtttga aattccagag ccagaaccta ctgacgcaga caaattggca
     2101 atagagaaag gcgagcagcc aatcagtgca gaccttaaaa gatttcgcaa ggaatatgtc
     2161 caaccagtac aacttaggat cttaaatgta tttcggcatt gggttgaaca tcatttttat
     2221 gactttgaaa gagacttgga attgcttgaa agactagaat ccttcatttc aagtgtaaga
     2281 gggaaagcta tgaaaaaatg ggtagagtca attgctaaga tcatcaggag gaagaagcaa
     2341 gctcaggcaa acggagtaag ccataatatt acctttgaaa gtccacctcc accaattgaa
     2401 tggcatatca gcaaaccagg acagtttgaa acatttgatc tcatgacact tcatccaata
     2461 gaaattgcac gtcagctgac acttttggag tctgatcttt acaggaaagt tcaaccgtct
     2521 gaacttgtag ggagtgtgtg gaccaaagaa gataaagaaa taaattctcc aaatttatta
     2581 aaaatgattc gccataccac aaatctcacc ctctggtttg aaaaatgcat tgtggaagca
     2641 gaaaattttg aagaacgggt ggcagtacta agtagaatta tagaaattct gcaagttttt
     2701 caagatttga ataatttcaa tggcgtattg gagatagtca gtgcagtaaa ttcagtgtca
     2761 gtatacagac tagaccatac ctttgaggca ctgcaggaaa ggaaaaggaa aattttggac
     2821 gaagctgtgg aattaagtca agatcacttt aaaaaatacc tagtaaaact taagtcaatc
     2881 aatccacctt gtgtgccttt ttttggaata tatttaacaa atattctgaa gaccgaagaa
     2941 gggaataatg attttttaaa aaagaaaggg aaagatttaa tcaatttcag taagaggagg
     3001 aaagtagctg aaattactgg agaaattcag cagtatcaga atcagcctta ctgtttacgg
     3061 atagaaccag atatgaggag attctttgaa aaccttaacc ccatgggaag tgcatctgaa
     3121 aaagagttta cagattattt gttcaacaag tcactagaaa ttgaacctcg aaactgcaaa
     3181 cagccacctc gatttcctag gaaatcaact ttttccttaa aatctcctgg aataaggcct
     3241 aacacaggcc gacatggctc taccccgcgg ggaagcggag ctactaactt cagcctgctg
     3301 aagcaggctg gagacgtgga ggagaaccct ggacctgcac cgggatccgg ggagtttctg
     3361 gcaaccacac tggaacggat cgagaaaaat ttcgtgatta ctgatccgag actgcctgac
     3421 aacccaatca tttttgcgag cgattccttc ctgcagctga cagaatattc tcgggaagag
     3481 atcctggggc gcaattgccg ttttctgcag ggacccgaga cagaccgtgc cactgttcgg
     3541 aaaatcagag atgctattga caaccagact gaagtgaccg ttcagctgat caattatacc
     3601 aagagcggca agaagttctg gaacgtgttc cacctgcagc cgatgcgcga ttataagggc
     3661 gacgtccagt acttcattgg cgtgcagctg gatggcaccg aacgtcttca tggcgccgct
     3721 gagcgtgagg cggtctgcct gatcaaaaag acagcctttc agattgctga ggcagcgaac
     3781 gacgaaaatt actttggaag cgggagtggg agcaaaaaga agaaaaagaa gtcaaagaca
     3841 aagtgtgtaa ttatgtaggc ggccgcgact ctagatcata atcagccata ccacatttgt
     3901 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     3961 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     4021 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4081 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     4141 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     4201 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     4261 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     4321 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     4381 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     4441 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     4501 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     4561 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4621 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4681 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     4741 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4801 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4861 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4921 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4981 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     5041 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     5101 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     5161 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     5221 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     5281 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5341 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5401 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5461 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5521 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5581 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     5641 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     5701 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     5761 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     5821 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     5881 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     5941 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     6001 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     6061 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     6121 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     6181 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     6241 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     6301 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     6361 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6421 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6481 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6541 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6601 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6661 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6721 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6781 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6841 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6901 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6961 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7021 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7081 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcac
//
