LOCUS       sequence-341725-        6730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mCherry-NES-SspB-ITSN1-DHPH-P2A-iLIDcaax 
            (opto-Cdc42).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6730)
  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 6730)
  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-ITSN1-DHPH-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..6730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        94..397
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        398..601
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     552..572
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      640..649
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             646..1353
                     /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(791..809)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1059..1078
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1402..1731
                     /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             2851..2907
                     /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             2929..3366
                     /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    3551..3672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3588..3607)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3642..3661
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3679..4134)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3816..3837)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4028..4047
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4161..4265
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4267..4624
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4267..4458
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4290..4310)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4475..4610
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4659..5453
                     /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             4659..5453
                     /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(4713..4732)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5323..5342
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5641..5660)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5685..5732
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6061..6649
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6550..6569
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat caattacggg
       61 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      121 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      181 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      241 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      301 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      361 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      421 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      481 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      541 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      601 tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt gagcaagggc
      661 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
      721 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
      781 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
      841 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
      901 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
      961 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     1021 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     1081 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     1141 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     1201 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     1261 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     1321 cactccaccg gcggcatgga cgagctgtac aagttcggca ttgatctgag cggcctgacc
     1381 ctgggtacca gtgctggagg tagctccccg aaacgcccta agctgctgcg tgaatattac
     1441 gattggctgg ttgataacag ctttacccca tatctggtgg tggatgccac atacctgggc
     1501 gtgaacgtgc ccgtggagta tgtgaaagac ggtcagatcg tgctgaatct gtctgcaagt
     1561 gcgaccggca acctgcaact gacaaatgat tttatccagt tcaacgcccg ctttaagggc
     1621 gtgtctcgtg aactgtatat cccgatgggt gccgctctgg ccatttacgc tcgcgagaac
     1681 ggcgatggtg tgatgttcga accagaagaa atctatgacg agctgaatat tggtctcgac
     1741 agtgctggag gtagtgctgg aggttcagct ggtggactcg agatgttgac cccaactgaa
     1801 agaaagcgac aaggatacat ccacgagctc attgtcaccg aggagaacta tgtgaatgac
     1861 ctgcagctgg tcacagagat ttttcaaaaa cccctgatgg agtctgagct gctgacagaa
     1921 aaagaggttg ctatgatttt tgtgaactgg aaggagctga ttatgtgtaa tatcaaacta
     1981 ctaaaagcgc tgagagtccg caagaagatg tccggggaga agatgcctgt gaagatgatt
     2041 ggagacatcc tgagcgcaca gctgccgcac atgcagccct acatccgctt ctgcagccgc
     2101 cagctcaacg gggctgccct gatccagcag aagacggatg aggccccaga cttcaaggag
     2161 ttcgtcaaaa gattggcaat ggatcctcgg tgtaaaggga tgccactctc tagttttata
     2221 ctgaagccta tgcaacgggt aacaagatac ccactgatca ttaaaaatat cctggaaaac
     2281 acccctgaaa accacccgga ccacagccac ttgaagcacg ccctggagaa ggcggaagag
     2341 ctctgttccc aggtgaacga aggggtgcgg gagaaggaga actctgaccg gctggagtgg
     2401 atccaggccc acgtgcagtg tgaaggcctg tctgagcaac ttgtgttcaa ttcagtgacc
     2461 aattgcttgg ggccgcgcaa atttctgcac agtgggaagc tctacaaggc caagagcaac
     2521 aaggagctgt atggcttcct tttcaacgac ttcctcctgc tgactcagat cacgaagcct
     2581 ttggggtctt ctggcaccga caaagtcttc agccccaaat caaacctgca gtataaaatg
     2641 tataaaacac ctattttcct aaatgaggtt ctagtaaaat tacccaccga cccttctgga
     2701 gacgagccca tcttccacat ctcccacatt gaccgcgtct atactctccg agcagaaagc
     2761 ataaatgaaa ggactgcctg ggtgcagaaa atcaaagctg cttctgaact ctacatagag
     2821 actgagaaaa agaagccgcg gggaagcgga gctactaact tcagcctgct gaagcaggct
     2881 ggagacgtgg aggagaaccc tggacctgca ccgggatccg gggagtttct ggcaaccaca
     2941 ctggaacgga tcgagaaaaa tttcgtgatt actgatccga gactgcctga caacccaatc
     3001 atttttgcga gcgattcctt cctgcagctg acagaatatt ctcgggaaga gatcctgggg
     3061 cgcaattgcc gttttctgca gggacccgag acagaccgtg ccactgttcg gaaaatcaga
     3121 gatgctattg acaaccagac tgaagtgacc gttcagctga tcaattatac caagagcggc
     3181 aagaagttct ggaacgtgtt ccacctgcag ccgatgcgcg attataaggg cgacgtccag
     3241 tacttcattg gcgtgcagct ggatggcacc gaacgtcttc atggcgccgc tgagcgtgag
     3301 gcggtctgcc tgatcaaaaa gacagccttt cagattgctg aggcagcgaa cgacgaaaat
     3361 tactttggaa gcgggagtgg gagcaaaaag aagaaaaaga agtcaaagac aaagtgtgta
     3421 attatgtagg cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
     3481 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     3541 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3601 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3661 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     3721 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3781 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3841 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3901 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3961 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     4021 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     4081 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     4141 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4201 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4261 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     4321 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     4381 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4441 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4501 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4561 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4621 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     4681 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     4741 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     4801 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     4861 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     4921 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     4981 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     5041 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     5101 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     5161 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     5221 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     5281 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     5341 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     5401 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     5461 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     5521 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     5581 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     5641 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     5701 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     5761 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     5821 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     5881 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     5941 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6001 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6061 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6121 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6181 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6241 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6301 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6361 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6421 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6481 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6541 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6601 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6661 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6721 gttatcccct
//
