LOCUS       sequence-341729-        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        107..410
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        411..614
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     565..585
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      653..662
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(659..661,662..664,665..1375)
                     /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(704..725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(965..984)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1312..1333
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1424..1753
                     /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             3374..3430
                     /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             3452..3889
                     /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    4074..4195
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4111..4130)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4165..4184
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4202..4657)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4339..4360)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4551..4570
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4684..4788
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4790..5147
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4790..4981
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4813..4833)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4998..5133
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5182..5976
                     /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             5182..5976
                     /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(5236..5255)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5846..5865
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6164..6183)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6208..6255
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6584..7172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7073..7092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt
       61 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      121 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      181 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      241 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      301 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      361 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      421 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      481 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      541 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      601 atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg tcgccaccat
      661 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      721 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      781 caagctgacc ctgaagttca tcagcaccac cggcaagctg cccgtgccct ggcccaccct
      841 cgtgaccacc ctcggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca
      901 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
      961 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1021 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1081 gctggagtac aactacaaca gccacaacgt ctatatcacc gccgacaagc agaagaacgg
     1141 catcaaggca aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga
     1201 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1261 cctgagctac cagtccaaac tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1321 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagttcgg
     1381 cattgatctg agcggcctga ccctgggtac cagtgctgga ggtagctccc cgaaacgccc
     1441 taagctgctg cgtgaatatt acgattggct ggttgataac agctttaccc catatctggt
     1501 ggtggatgcc acatacctgg gcgtgaacgt gcccgtggag tatgtgaaag acggtcagat
     1561 cgtgctgaat ctgtctgcaa gtgcgaccgg caacctgcaa ctgacaaatg attttatcca
     1621 gttcaacgcc cgctttaagg gcgtgtctcg tgaactgtat atcccgatgg gtgccgctct
     1681 ggccatttac gctcgcgaga acggcgatgg tgtgatgttc gaaccagaag aaatctatga
     1741 cgagctgaat attggtctcg acagtgctgg aggtagtgct ggaggttcag ctggtggact
     1801 cgagatgctc ccgcggcccc tgcggctgct tttggacacg agcccccccg ggggagtcgt
     1861 actgagcagc ttccgaagcc gggaccccga agagggtggg ggcccaggtg gcctggtcgt
     1921 gggcgggggg caggaggaag aggaggagga agaagaagag gcccctgtgt ccgtctggga
     1981 tgaggaggag gatggtgccg tgtttaccgt cacaagccgc caatatcgac ctcttgatcc
     2041 cttggtccct atgcctcccc cacgttcctc ccgacggctc cgagctggca ctctggaggc
     2101 cctggtcaga cacctactgg atacccggac atcagggact gatgtgagct tcatgtcagc
     2161 cttcctggct acccaccggg ccttcacctc cacgcctgcc ttgctagggc ttatggctga
     2221 caggctggaa gcccttgaat cccatcctac cgacgaacta gagaggacaa cagaggtagc
     2281 catctctgta ctgtcaacct ggctggcctc tcaccctgag gattttggct ctgaggccaa
     2341 gggtcagctt gaccggcttg agagcttctt acttcagaca gggtatgcag cagggaaggg
     2401 tgttgggggg ggcagcgctg acctcatccg caatctccgg tcccgggtgg acccccaggc
     2461 ccccgacctt cctaagcccc tggccctccc cggcgatccc cctgctgacc ccacggatgt
     2521 cctggtgttc ctcgctgacc acttggccga acagctgacc ctgctagatg cggaactttt
     2581 tctcaatttg atcccctctc agtgcctggg aggcctgtgg ggtcacagag accggccagg
     2641 acattctcac ctctgcccat ctgtccgagc tactgtcaca cagtttaaca aggtggcagg
     2701 ggcagtggtt agttctgtcc tgggggctac ttccactgga gagggacctg gggaggtgac
     2761 catacggcca ctccgtcccc cacagagggc ccggctcctg gagaagtgga tccgcgtggc
     2821 agaggagtgc cggctgctcc gaaacttctc ttcagtttat gccgtggtgt cagccctgca
     2881 gtccagcccc atccacaggc ttcgggcagc ctggggggaa gcaaccaggg acagcctcag
     2941 agtcttttcc agcctctgcc agattttctc cgaggaggat aattattccc agagtcggga
     3001 gctgctcgtg caggaggtga agctgcagtc tcctctggag ccacactcca agaaggcccc
     3061 gaggtctggc tcccggggtg ggggtgtggt cccatacctt ggcaccttcc tgaaggacct
     3121 tgtgatgctg gatgcagcct ccaaggatga gttggagaat ggatacatca attttgacaa
     3181 gcggaggaag gagtttgcag tcctttctga gttgcgacgg ctccagaatg aatgtcgtgg
     3241 ctataacctc caacctgacc atgatatcca gaggtggcta caggggctcc ggccactgac
     3301 agaggctcag agccatcgtg tatcctgtga ggtggagcca cctggttcca gtgaccctcc
     3361 gcggggaagc ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa
     3421 ccctggacct gcaccgggat ccggggagtt tctggcaacc acactggaac ggatcgagaa
     3481 aaatttcgtg attactgatc cgagactgcc tgacaaccca atcatttttg cgagcgattc
     3541 cttcctgcag ctgacagaat attctcggga agagatcctg gggcgcaatt gccgttttct
     3601 gcagggaccc gagacagacc gtgccactgt tcggaaaatc agagatgcta ttgacaacca
     3661 gactgaagtg accgttcagc tgatcaatta taccaagagc ggcaagaagt tctggaacgt
     3721 gttccacctg cagccgatgc gcgattataa gggcgacgtc cagtacttca ttggcgtgca
     3781 gctggatggc accgaacgtc ttcatggcgc cgctgagcgt gaggcggtct gcctgatcaa
     3841 aaagacagcc tttcagattg ctgaggcagc gaacgacgaa aattactttg gaagcgggag
     3901 tgggagcaaa aagaagaaaa agaagtcaaa gacaaagtgt gtaattatgt aggcggccgc
     3961 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     4021 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     4081 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4141 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
     4201 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     4261 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     4321 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     4381 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     4441 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     4501 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     4561 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     4621 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     4681 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4741 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     4801 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     4861 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     4921 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     4981 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     5041 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     5101 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     5161 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     5221 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5281 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5341 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     5401 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5461 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5521 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5581 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5641 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5701 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5761 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5821 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5881 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5941 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     6001 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     6061 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     6121 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     6181 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     6241 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     6301 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     6361 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     6421 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6481 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6541 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6601 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6661 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6721 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6781 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6841 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6901 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6961 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7021 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7081 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7141 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7201 tcctggcctt ttgctggcct tttgctcaca tgttctttcc
//
