LOCUS       sequence-341719-        6340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mCherry-NES-SspB-LARG-DH-P2A-iLIDcaax 
            (opto-RhoA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6340)
  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 6340)
  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-LARG-DH-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..6340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        122..425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        426..629
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     580..600
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      668..677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             674..1381
                     /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(819..837)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1087..1106
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1430..1759
                     /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             2489..2545
                     /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             2567..3004
                     /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    3189..3310
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3226..3245)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3280..3299
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3317..3772)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3454..3475)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3666..3685
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3799..3903
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3905..4262
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3905..4096
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3928..3948)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4113..4248
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4297..5091
                     /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             4297..5091
                     /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(4351..4370)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4961..4980
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5279..5298)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5323..5370
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5699..6287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6188..6207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
       61 ttagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      121 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      181 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      241 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      301 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      361 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      421 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      481 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      541 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      601 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct
      661 accggtcgcc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
      721 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
      781 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
      841 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
      901 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
      961 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     1021 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     1081 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     1141 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     1201 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     1261 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     1321 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     1381 gttcggcatt gatctgagcg gcctgaccct gggtaccagt gctggaggta gctccccgaa
     1441 acgccctaag ctgctgcgtg aatattacga ttggctggtt gataacagct ttaccccata
     1501 tctggtggtg gatgccacat acctgggcgt gaacgtgccc gtggagtatg tgaaagacgg
     1561 tcagatcgtg ctgaatctgt ctgcaagtgc gaccggcaac ctgcaactga caaatgattt
     1621 tatccagttc aacgcccgct ttaagggcgt gtctcgtgaa ctgtatatcc cgatgggtgc
     1681 cgctctggcc atttacgctc gcgagaacgg cgatggtgtg atgttcgaac cagaagaaat
     1741 ctatgacgag ctgaatattg gtctcgacag tgctggaggt agtgctggag gttcagctgg
     1801 tggactcgag ccacccaact ggcagcagct tgttagtcga gaagtgttac tgggactaaa
     1861 accttgtgaa atcaaaagac aggaagtgat taatgaattg ttctacactg aaagagctca
     1921 tgttcgaaca ctgaaggttc ttgatcaagt gttctatcag cgagtatcca gagaaggaat
     1981 tctgtcaccc tcagagctac ggaaaatttt ttcaaacttg gaagatattc ttcaacttca
     2041 tattggattg aatgaacaaa tgaaggctgt tcgaaagaga aatgagacct ctgttatcga
     2101 tcagattggg gaagatttgc tgacatggtt cagcggacca ggagaggaga aattgaaaca
     2161 tgctgctgct accttttgca gtaaccaacc tttcgccctg gaaatgatca aatctcgtca
     2221 gaaaaaggat tctcgatttc agacttttgt gcaagatgct gaaagtaatc cactgtgtcg
     2281 tcgtcttcaa ctgaaggata ttattcccac tcaaatgcaa aggcttacta agtacccact
     2341 tctgttggat aatattgcca aatacacaga atggccaaca gaaagggaga aggtgaagaa
     2401 agctgcagat cactgtcgtc agatcttaaa ttatgtaaat caggctgtca aggaggcaga
     2461 aaacaagcag cgcccgcggg gaagcggagc tactaacttc agcctgctga agcaggctgg
     2521 agacgtggag gagaaccctg gacctgcacc gggatccggg gagtttctgg caaccacact
     2581 ggaacggatc gagaaaaatt tcgtgattac tgatccgaga ctgcctgaca acccaatcat
     2641 ttttgcgagc gattccttcc tgcagctgac agaatattct cgggaagaga tcctggggcg
     2701 caattgccgt tttctgcagg gacccgagac agaccgtgcc actgttcgga aaatcagaga
     2761 tgctattgac aaccagactg aagtgaccgt tcagctgatc aattatacca agagcggcaa
     2821 gaagttctgg aacgtgttcc acctgcagcc gatgcgcgat tataagggcg acgtccagta
     2881 cttcattggc gtgcagctgg atggcaccga acgtcttcat ggcgccgctg agcgtgaggc
     2941 ggtctgcctg atcaaaaaga cagcctttca gattgctgag gcagcgaacg acgaaaatta
     3001 ctttggaagc gggagtggga gcaaaaagaa gaaaaagaag tcaaagacaa agtgtgtaat
     3061 tatgtaggcg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac
     3121 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     3181 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3241 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3301 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     3361 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     3421 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     3481 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     3541 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     3601 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     3661 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     3721 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     3781 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3841 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3901 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3961 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     4021 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4081 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4141 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4201 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4261 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4321 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4381 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4441 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     4501 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4561 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4621 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4681 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4741 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4801 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     4861 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4921 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4981 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5041 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5101 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     5161 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5221 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     5281 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     5341 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     5401 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     5461 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     5521 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     5581 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5641 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5701 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5761 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5821 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5881 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5941 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6001 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6061 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6121 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6181 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6241 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6301 cggccttttt acggttcctg gccttttgct ggccttttgc
//
