LOCUS       sequence-341718-        5701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mCherry-NES-SspB-P2A-iLIDcaax (opto-control)
            containing multiple cloning sites (MCS).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5701)
  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 5701)
  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-MCS-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..5701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        136..439
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        440..643
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     594..614
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      682..691
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             688..1395
                     /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(833..851)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1101..1120
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1444..1773
                     /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             1864..1920
                     /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             1942..2379
                     /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    2564..2685
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2601..2620)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2655..2674
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2692..3147)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2829..2850)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3041..3060
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3174..3278
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3280..3637
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3280..3471
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3303..3323)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3488..3623
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3672..4466
                     /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             3672..4466
                     /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(3726..3745)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4336..4355
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4654..4673)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4698..4745
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5074..5662
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5563..5582
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
       61 attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc
      121 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      181 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      481 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      541 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      601 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
      661 gatccgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagga taacatggcc
      721 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
      781 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
      841 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
      901 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
      961 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1021 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1081 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1141 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     1201 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     1261 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     1321 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     1381 gacgagctgt acaagttcgg cattgatctg agcggcctga ccctgggtac cagtgctgga
     1441 ggtagctccc cgaaacgccc taagctgctg cgtgaatatt acgattggct ggttgataac
     1501 agctttaccc catatctggt ggtggatgcc acatacctgg gcgtgaacgt gcccgtggag
     1561 tatgtgaaag acggtcagat cgtgctgaat ctgtctgcaa gtgcgaccgg caacctgcaa
     1621 ctgacaaatg attttatcca gttcaacgcc cgctttaagg gcgtgtctcg tgaactgtat
     1681 atcccgatgg gtgccgctct ggccatttac gctcgcgaga acggcgatgg tgtgatgttc
     1741 gaaccagaag aaatctatga cgagctgaat attggtctcg acagtgctgg aggtagtgct
     1801 ggaggttcag ctggtggact cgaggtcgac gaattcgagc tcgatatccc gcggggaagc
     1861 ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct
     1921 gcaccgggat ccggggagtt tctggcaacc acactggaac ggatcgagaa aaatttcgtg
     1981 attactgatc cgagactgcc tgacaaccca atcatttttg cgagcgattc cttcctgcag
     2041 ctgacagaat attctcggga agagatcctg gggcgcaatt gccgttttct gcagggaccc
     2101 gagacagacc gtgccactgt tcggaaaatc agagatgcta ttgacaacca gactgaagtg
     2161 accgttcagc tgatcaatta taccaagagc ggcaagaagt tctggaacgt gttccacctg
     2221 cagccgatgc gcgattataa gggcgacgtc cagtacttca ttggcgtgca gctggatggc
     2281 accgaacgtc ttcatggcgc cgctgagcgt gaggcggtct gcctgatcaa aaagacagcc
     2341 tttcagattg ctgaggcagc gaacgacgaa aattactttg gaagcgggag tgggagcaaa
     2401 aagaagaaaa agaagtcaaa gacaaagtgt gtaattatgt aggcggccgc gactctagat
     2461 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     2521 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     2581 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2641 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa
     2701 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2761 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2821 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2881 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2941 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3001 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     3061 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3121 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3181 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3241 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     3301 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3361 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3421 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3481 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3541 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3601 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     3661 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3721 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3781 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3841 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3901 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3961 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4021 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4081 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4141 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     4201 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4261 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4321 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4381 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4441 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4501 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4561 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4621 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     4681 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     4741 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     4801 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     4861 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     4921 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4981 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5041 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5101 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5161 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5221 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5281 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5341 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5401 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5461 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5521 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5581 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5641 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5701 t
//
