LOCUS       sequence-337217-        7240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mVenus-NES-SspB-RGL2-GEF-P2A-iLIDcaax 
            (opto-Ral).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7240)
  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 7240)
  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: mVenus-NES-SspB-RGL2-GEF-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..7240
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="photostable derivative of mVenus (Lee et al., 
                     2020)"
                     /label=mGold
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1378..1707
                     /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             3328..3384
                     /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             3406..3843
                     /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    4028..4149
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4065..4084)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4119..4138
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4156..4611)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4293..4314)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4505..4524
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4638..4742
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4744..5101
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4744..4935
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4767..4787)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4952..5087
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5136..5930
                     /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             5136..5930
                     /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(5190..5209)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5800..5819
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6118..6137)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6162..6209
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6538..7126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7027..7046
                     /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 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatcagca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctcggc tacggcctgc agtgcttcgc ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac
     1081 aagcagaaga acggcatcaa ggcaaacttc aagatccgcc acaacatcga ggacggcggc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag ctaccagtcc aaactgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt tcggcattga tctgagcggc ctgaccctgg gtaccagtgc tggaggtagc
     1381 tccccgaaac gccctaagct gctgcgtgaa tattacgatt ggctggttga taacagcttt
     1441 accccatatc tggtggtgga tgccacatac ctgggcgtga acgtgcccgt ggagtatgtg
     1501 aaagacggtc agatcgtgct gaatctgtct gcaagtgcga ccggcaacct gcaactgaca
     1561 aatgatttta tccagttcaa cgcccgcttt aagggcgtgt ctcgtgaact gtatatcccg
     1621 atgggtgccg ctctggccat ttacgctcgc gagaacggcg atggtgtgat gttcgaacca
     1681 gaagaaatct atgacgagct gaatattggt ctcgacagtg ctggaggtag tgctggaggt
     1741 tcagctggtg gactcgagat gctcccgcgg cccctgcggc tgcttttgga cacgagcccc
     1801 cccgggggag tcgtactgag cagcttccga agccgggacc ccgaagaggg tgggggccca
     1861 ggtggcctgg tcgtgggcgg ggggcaggag gaagaggagg aggaagaaga agaggcccct
     1921 gtgtccgtct gggatgagga ggaggatggt gccgtgttta ccgtcacaag ccgccaatat
     1981 cgacctcttg atcccttggt ccctatgcct cccccacgtt cctcccgacg gctccgagct
     2041 ggcactctgg aggccctggt cagacaccta ctggataccc ggacatcagg gactgatgtg
     2101 agcttcatgt cagccttcct ggctacccac cgggccttca cctccacgcc tgccttgcta
     2161 gggcttatgg ctgacaggct ggaagccctt gaatcccatc ctaccgacga actagagagg
     2221 acaacagagg tagccatctc tgtactgtca acctggctgg cctctcaccc tgaggatttt
     2281 ggctctgagg ccaagggtca gcttgaccgg cttgagagct tcttacttca gacagggtat
     2341 gcagcaggga agggtgttgg ggggggcagc gctgacctca tccgcaatct ccggtcccgg
     2401 gtggaccccc aggcccccga ccttcctaag cccctggccc tccccggcga tccccctgct
     2461 gaccccacgg atgtcctggt gttcctcgct gaccacttgg ccgaacagct gaccctgcta
     2521 gatgcggaac tttttctcaa tttgatcccc tctcagtgcc tgggaggcct gtggggtcac
     2581 agagaccggc caggacattc tcacctctgc ccatctgtcc gagctactgt cacacagttt
     2641 aacaaggtgg caggggcagt ggttagttct gtcctggggg ctacttccac tggagaggga
     2701 cctggggagg tgaccatacg gccactccgt cccccacaga gggcccggct cctggagaag
     2761 tggatccgcg tggcagagga gtgccggctg ctccgaaact tctcttcagt ttatgccgtg
     2821 gtgtcagccc tgcagtccag ccccatccac aggcttcggg cagcctgggg ggaagcaacc
     2881 agggacagcc tcagagtctt ttccagcctc tgccagattt tctccgagga ggataattat
     2941 tcccagagtc gggagctgct cgtgcaggag gtgaagctgc agtctcctct ggagccacac
     3001 tccaagaagg ccccgaggtc tggctcccgg ggtgggggtg tggtcccata ccttggcacc
     3061 ttcctgaagg accttgtgat gctggatgca gcctccaagg atgagttgga gaatggatac
     3121 atcaattttg acaagcggag gaaggagttt gcagtccttt ctgagttgcg acggctccag
     3181 aatgaatgtc gtggctataa cctccaacct gaccatgata tccagaggtg gctacagggg
     3241 ctccggccac tgacagaggc tcagagccat cgtgtatcct gtgaggtgga gccacctggt
     3301 tccagtgacc ctccgcgggg aagcggagct actaacttca gcctgctgaa gcaggctgga
     3361 gacgtggagg agaaccctgg acctgcaccg ggatccgggg agtttctggc aaccacactg
     3421 gaacggatcg agaaaaattt cgtgattact gatccgagac tgcctgacaa cccaatcatt
     3481 tttgcgagcg attccttcct gcagctgaca gaatattctc gggaagagat cctggggcgc
     3541 aattgccgtt ttctgcaggg acccgagaca gaccgtgcca ctgttcggaa aatcagagat
     3601 gctattgaca accagactga agtgaccgtt cagctgatca attataccaa gagcggcaag
     3661 aagttctgga acgtgttcca cctgcagccg atgcgcgatt ataagggcga cgtccagtac
     3721 ttcattggcg tgcagctgga tggcaccgaa cgtcttcatg gcgccgctga gcgtgaggcg
     3781 gtctgcctga tcaaaaagac agcctttcag attgctgagg cagcgaacga cgaaaattac
     3841 tttggaagcg ggagtgggag caaaaagaag aaaaagaagt caaagacaaa gtgtgtaatt
     3901 atgtaggcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     3961 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     4021 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4081 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4141 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     4201 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     4261 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     4321 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     4381 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     4441 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     4501 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     4561 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     4621 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4681 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4741 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     4801 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     4861 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4921 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4981 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     5041 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     5101 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     5161 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     5221 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     5281 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     5341 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     5401 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     5461 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     5521 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     5581 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     5641 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     5701 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     5761 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     5821 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     5881 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     5941 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     6001 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     6061 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
     6121 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     6181 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
     6241 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
     6301 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
     6361 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt
     6421 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6481 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6541 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6601 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6661 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     6721 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6781 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6841 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6901 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6961 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7021 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7081 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7141 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     7201 atcccctgat tctgtggata accgtattac cgccatgcat
//
