LOCUS       sequence-341728-        6850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mVenus-NES-SspB-C3Gcat-P2A-iLIDcaax 
            (opto-Rap).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6850)
  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 6850)
  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-C3Gcat-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..6850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        90..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      636..645
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             642..1349
                     /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(787..805)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1055..1074
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1398..1727
                     /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             2967..3023
                     /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             3045..3482
                     /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    3667..3788
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3704..3723)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3758..3777
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3795..4250)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3932..3953)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4144..4163
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4277..4381
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4383..4740
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4383..4574
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4406..4426)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4591..4726
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4775..5569
                     /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             4775..5569
                     /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(4829..4848)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5439..5458
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5757..5776)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5801..5848
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6177..6765
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6666..6685
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgtggataa ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
      601 ttagtgaacc gtcagatccg ctagcgctac cggtcgccac catggtgagc aagggcgagg
      661 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
      721 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
      781 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
      841 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
      901 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
      961 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     1021 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     1081 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     1141 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     1201 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     1261 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     1321 ccaccggcgg catggacgag ctgtacaagt tcggcattga tctgagcggc ctgaccctgg
     1381 gtaccagtgc tggaggtagc tccccgaaac gccctaagct gctgcgtgaa tattacgatt
     1441 ggctggttga taacagcttt accccatatc tggtggtgga tgccacatac ctgggcgtga
     1501 acgtgcccgt ggagtatgtg aaagacggtc agatcgtgct gaatctgtct gcaagtgcga
     1561 ccggcaacct gcaactgaca aatgatttta tccagttcaa cgcccgcttt aagggcgtgt
     1621 ctcgtgaact gtatatcccg atgggtgccg ctctggccat ttacgctcgc gagaacggcg
     1681 atggtgtgat gttcgaacca gaagaaatct atgacgagct gaatattggt ctcgacagtg
     1741 ctggaggtag tgctggaggt tcagctggtg gactcgagga tgacgggccg gacgtccgcg
     1801 gaggatctgg ggacatctta ctggtccatg ctactgagac tgacaggaaa gatttggtgt
     1861 tgtactgcga ggcattcctg accacctaca ggaccttcat ctccccagag gagctcatca
     1921 agaagctgca gtacagatat gagaaattct ctccctttgc cgacacattc aagaagcgcg
     1981 tcagcaagaa cacgttcttc gtgctggtac gggtggtgga tgagctctgc ctggtggagt
     2041 tgacagaaga gatcctgaag ctgctgatgg aactggtctt ccgcctggtg tgcaatgggg
     2101 agctgagcct ggcccgtgtg ctccggaaga acatcctgga caaggtggac cagaagaagc
     2161 tactcaggtg tgccacctcc agccagcccc tggcagcccg gggggtagca gccaggccgg
     2221 ggaccttgca cgactttcac agccatgaga tagcggagca gctaacgctg ctggatgctg
     2281 agctcttcta taaaatagag attcctgagg ttttgctttg ggcaaaagag cagaatgagg
     2341 agaagagccc caacttgacc cagttcacgg agcacttcaa caacatgtcc tactgggtcc
     2401 ggtccataat catgttacag gaaaaggccc aggacaggga acggctgctc ttgaagttca
     2461 tcaagatcat gaagcacttg cggaagctga ataacttcaa ctcctacttg gccatcctct
     2521 ctgccctgga ctcggcgccc atccgcaggc tggagtggca gaagcagact tcagagggcc
     2581 tggccgagta ctgcacactg atcgacagct cgtcctcctt ccgagcctac cgggccgccc
     2641 tctcggaggt ggaaccgccg tgcatcccgt acctggggct gatcctgcag gacctgacct
     2701 tcgttcacct gggaaaccca gactacatcg acgggaaagt gaacttctcc aagcggtggc
     2761 agcagttcaa catcctcgac agcatgcgct gcttccagca ggcgcactat gacatgcgga
     2821 ggaacgacga cattataaac ttcttcaatg acttcagtga ccacctggct gaggaggccc
     2881 tatgggaact gtctctgaaa attaaaccca ggaacataac aaggagaaaa acagaccggg
     2941 aagagaagac cccgcgggga agcggagcta ctaacttcag cctgctgaag caggctggag
     3001 acgtggagga gaaccctgga cctgcaccgg gatccgggga gtttctggca accacactgg
     3061 aacggatcga gaaaaatttc gtgattactg atccgagact gcctgacaac ccaatcattt
     3121 ttgcgagcga ttccttcctg cagctgacag aatattctcg ggaagagatc ctggggcgca
     3181 attgccgttt tctgcaggga cccgagacag accgtgccac tgttcggaaa atcagagatg
     3241 ctattgacaa ccagactgaa gtgaccgttc agctgatcaa ttataccaag agcggcaaga
     3301 agttctggaa cgtgttccac ctgcagccga tgcgcgatta taagggcgac gtccagtact
     3361 tcattggcgt gcagctggat ggcaccgaac gtcttcatgg cgccgctgag cgtgaggcgg
     3421 tctgcctgat caaaaagaca gcctttcaga ttgctgaggc agcgaacgac gaaaattact
     3481 ttggaagcgg gagtgggagc aaaaagaaga aaaagaagtc aaagacaaag tgtgtaatta
     3541 tgtaggcggc cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt
     3601 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3661 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3721 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3781 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     3841 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     3901 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     3961 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     4021 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     4081 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     4141 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     4201 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     4261 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4321 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4381 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     4441 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4501 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4561 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4621 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4681 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4741 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     4801 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     4861 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     4921 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     4981 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     5041 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     5101 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     5161 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     5221 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     5281 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     5341 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     5401 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     5461 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     5521 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     5581 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     5641 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5701 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     5761 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     5821 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     5881 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     5941 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     6001 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     6061 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6121 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6181 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6241 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6301 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6361 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6421 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6481 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6541 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6601 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6661 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6721 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6781 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6841 tcccctgatt
//
