LOCUS       sequence-341731-        7231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mCherry-NES-SspB-RGL2-GEF-P2A-iLIDcaax 
            (opto-Ral).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7231)
  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 7231)
  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-RGL2-GEF-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..7231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        71..374
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        375..578
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     529..549
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      617..626
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             623..1330
                     /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(768..786)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1036..1055
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1379..1708
                     /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             3329..3385
                     /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             3407..3844
                     /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    4029..4150
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4066..4085)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4120..4139
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4157..4612)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4294..4315)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4506..4525
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4639..4743
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4745..5102
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4745..4936
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4768..4788)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4953..5088
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5137..5931
                     /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             5137..5931
                     /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(5191..5210)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5801..5820
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6119..6138)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6163..6210
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6539..7127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7028..7047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
       61 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      121 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      181 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      241 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      301 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      361 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      421 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      481 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      541 gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc
      601 gctagcgcta ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat
      661 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
      721 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
      781 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
      841 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
      901 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
      961 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     1021 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
     1081 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     1141 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     1201 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     1261 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     1321 gctgtacaag ttcggcattg atctgagcgg cctgaccctg ggtaccagtg ctggaggtag
     1381 ctccccgaaa cgccctaagc tgctgcgtga atattacgat tggctggttg ataacagctt
     1441 taccccatat ctggtggtgg atgccacata cctgggcgtg aacgtgcccg tggagtatgt
     1501 gaaagacggt cagatcgtgc tgaatctgtc tgcaagtgcg accggcaacc tgcaactgac
     1561 aaatgatttt atccagttca acgcccgctt taagggcgtg tctcgtgaac tgtatatccc
     1621 gatgggtgcc gctctggcca tttacgctcg cgagaacggc gatggtgtga tgttcgaacc
     1681 agaagaaatc tatgacgagc tgaatattgg tctcgacagt gctggaggta gtgctggagg
     1741 ttcagctggt ggactcgaga tgctcccgcg gcccctgcgg ctgcttttgg acacgagccc
     1801 ccccggggga gtcgtactga gcagcttccg aagccgggac cccgaagagg gtgggggccc
     1861 aggtggcctg gtcgtgggcg gggggcagga ggaagaggag gaggaagaag aagaggcccc
     1921 tgtgtccgtc tgggatgagg aggaggatgg tgccgtgttt accgtcacaa gccgccaata
     1981 tcgacctctt gatcccttgg tccctatgcc tcccccacgt tcctcccgac ggctccgagc
     2041 tggcactctg gaggccctgg tcagacacct actggatacc cggacatcag ggactgatgt
     2101 gagcttcatg tcagccttcc tggctaccca ccgggccttc acctccacgc ctgccttgct
     2161 agggcttatg gctgacaggc tggaagccct tgaatcccat cctaccgacg aactagagag
     2221 gacaacagag gtagccatct ctgtactgtc aacctggctg gcctctcacc ctgaggattt
     2281 tggctctgag gccaagggtc agcttgaccg gcttgagagc ttcttacttc agacagggta
     2341 tgcagcaggg aagggtgttg gggggggcag cgctgacctc atccgcaatc tccggtcccg
     2401 ggtggacccc caggcccccg accttcctaa gcccctggcc ctccccggcg atccccctgc
     2461 tgaccccacg gatgtcctgg tgttcctcgc tgaccacttg gccgaacagc tgaccctgct
     2521 agatgcggaa ctttttctca atttgatccc ctctcagtgc ctgggaggcc tgtggggtca
     2581 cagagaccgg ccaggacatt ctcacctctg cccatctgtc cgagctactg tcacacagtt
     2641 taacaaggtg gcaggggcag tggttagttc tgtcctgggg gctacttcca ctggagaggg
     2701 acctggggag gtgaccatac ggccactccg tcccccacag agggcccggc tcctggagaa
     2761 gtggatccgc gtggcagagg agtgccggct gctccgaaac ttctcttcag tttatgccgt
     2821 ggtgtcagcc ctgcagtcca gccccatcca caggcttcgg gcagcctggg gggaagcaac
     2881 cagggacagc ctcagagtct tttccagcct ctgccagatt ttctccgagg aggataatta
     2941 ttcccagagt cgggagctgc tcgtgcagga ggtgaagctg cagtctcctc tggagccaca
     3001 ctccaagaag gccccgaggt ctggctcccg gggtgggggt gtggtcccat accttggcac
     3061 cttcctgaag gaccttgtga tgctggatgc agcctccaag gatgagttgg agaatggata
     3121 catcaatttt gacaagcgga ggaaggagtt tgcagtcctt tctgagttgc gacggctcca
     3181 gaatgaatgt cgtggctata acctccaacc tgaccatgat atccagaggt ggctacaggg
     3241 gctccggcca ctgacagagg ctcagagcca tcgtgtatcc tgtgaggtgg agccacctgg
     3301 ttccagtgac cctccgcggg gaagcggagc tactaacttc agcctgctga agcaggctgg
     3361 agacgtggag gagaaccctg gacctgcacc gggatccggg gagtttctgg caaccacact
     3421 ggaacggatc gagaaaaatt tcgtgattac tgatccgaga ctgcctgaca acccaatcat
     3481 ttttgcgagc gattccttcc tgcagctgac agaatattct cgggaagaga tcctggggcg
     3541 caattgccgt tttctgcagg gacccgagac agaccgtgcc actgttcgga aaatcagaga
     3601 tgctattgac aaccagactg aagtgaccgt tcagctgatc aattatacca agagcggcaa
     3661 gaagttctgg aacgtgttcc acctgcagcc gatgcgcgat tataagggcg acgtccagta
     3721 cttcattggc gtgcagctgg atggcaccga acgtcttcat ggcgccgctg agcgtgaggc
     3781 ggtctgcctg atcaaaaaga cagcctttca gattgctgag gcagcgaacg acgaaaatta
     3841 ctttggaagc gggagtggga gcaaaaagaa gaaaaagaag tcaaagacaa agtgtgtaat
     3901 tatgtaggcg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac
     3961 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     4021 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4081 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4141 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     4201 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     4261 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     4321 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     4381 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     4441 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     4501 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     4561 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     4621 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4681 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4741 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     4801 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     4861 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4921 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4981 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     5041 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5101 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     5161 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     5221 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     5281 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     5341 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     5401 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     5461 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     5521 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     5581 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     5641 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     5701 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     5761 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     5821 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5881 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5941 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     6001 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     6061 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     6121 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     6181 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     6241 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     6301 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     6361 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     6421 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6481 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6541 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6601 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6661 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6721 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6781 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6841 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6901 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6961 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7021 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7081 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7141 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7201 tatcccctga ttctgtggat aaccgtatta c
//
