LOCUS       sequence-341717-        6859 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 6859)
  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 6859)
  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-C3Gcat-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..6859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        84..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        388..591
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     542..562
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      630..639
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(636..638,639..641,642..1352)
                     /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(681..702)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(942..961)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1289..1310
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1401..1730
                     /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             2970..3026
                     /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             3048..3485
                     /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    3670..3791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3707..3726)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3761..3780
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3798..4253)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3935..3956)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4147..4166
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4280..4384
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4386..4743
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4386..4577
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4409..4429)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4594..4729
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4778..5572
                     /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             4778..5572
                     /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(4832..4851)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5442..5461
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5760..5779)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5804..5851
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6180..6768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6669..6688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ataaccgtat taccgccatg cattagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      421 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      481 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      541 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg
      601 aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt gagcaagggc gaggagctgt
      661 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
      721 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatca
      781 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctc ggctacggcc
      841 tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
      901 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
      961 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1021 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1081 acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggcaaac ttcaagatcc
     1141 gccacaacat cgaggacggc ggcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1201 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag tccaaactga
     1261 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1321 ggatcactct cggcatggac gagctgtaca agttcggcat tgatctgagc ggcctgaccc
     1381 tgggtaccag tgctggaggt agctccccga aacgccctaa gctgctgcgt gaatattacg
     1441 attggctggt tgataacagc tttaccccat atctggtggt ggatgccaca tacctgggcg
     1501 tgaacgtgcc cgtggagtat gtgaaagacg gtcagatcgt gctgaatctg tctgcaagtg
     1561 cgaccggcaa cctgcaactg acaaatgatt ttatccagtt caacgcccgc tttaagggcg
     1621 tgtctcgtga actgtatatc ccgatgggtg ccgctctggc catttacgct cgcgagaacg
     1681 gcgatggtgt gatgttcgaa ccagaagaaa tctatgacga gctgaatatt ggtctcgaca
     1741 gtgctggagg tagtgctgga ggttcagctg gtggactcga ggatgacggg ccggacgtcc
     1801 gcggaggatc tggggacatc ttactggtcc atgctactga gactgacagg aaagatttgg
     1861 tgttgtactg cgaggcattc ctgaccacct acaggacctt catctcccca gaggagctca
     1921 tcaagaagct gcagtacaga tatgagaaat tctctccctt tgccgacaca ttcaagaagc
     1981 gcgtcagcaa gaacacgttc ttcgtgctgg tacgggtggt ggatgagctc tgcctggtgg
     2041 agttgacaga agagatcctg aagctgctga tggaactggt cttccgcctg gtgtgcaatg
     2101 gggagctgag cctggcccgt gtgctccgga agaacatcct ggacaaggtg gaccagaaga
     2161 agctactcag gtgtgccacc tccagccagc ccctggcagc ccggggggta gcagccaggc
     2221 cggggacctt gcacgacttt cacagccatg agatagcgga gcagctaacg ctgctggatg
     2281 ctgagctctt ctataaaata gagattcctg aggttttgct ttgggcaaaa gagcagaatg
     2341 aggagaagag ccccaacttg acccagttca cggagcactt caacaacatg tcctactggg
     2401 tccggtccat aatcatgtta caggaaaagg cccaggacag ggaacggctg ctcttgaagt
     2461 tcatcaagat catgaagcac ttgcggaagc tgaataactt caactcctac ttggccatcc
     2521 tctctgccct ggactcggcg cccatccgca ggctggagtg gcagaagcag acttcagagg
     2581 gcctggccga gtactgcaca ctgatcgaca gctcgtcctc cttccgagcc taccgggccg
     2641 ccctctcgga ggtggaaccg ccgtgcatcc cgtacctggg gctgatcctg caggacctga
     2701 ccttcgttca cctgggaaac ccagactaca tcgacgggaa agtgaacttc tccaagcggt
     2761 ggcagcagtt caacatcctc gacagcatgc gctgcttcca gcaggcgcac tatgacatgc
     2821 ggaggaacga cgacattata aacttcttca atgacttcag tgaccacctg gctgaggagg
     2881 ccctatggga actgtctctg aaaattaaac ccaggaacat aacaaggaga aaaacagacc
     2941 gggaagagaa gaccccgcgg ggaagcggag ctactaactt cagcctgctg aagcaggctg
     3001 gagacgtgga ggagaaccct ggacctgcac cgggatccgg ggagtttctg gcaaccacac
     3061 tggaacggat cgagaaaaat ttcgtgatta ctgatccgag actgcctgac aacccaatca
     3121 tttttgcgag cgattccttc ctgcagctga cagaatattc tcgggaagag atcctggggc
     3181 gcaattgccg ttttctgcag ggacccgaga cagaccgtgc cactgttcgg aaaatcagag
     3241 atgctattga caaccagact gaagtgaccg ttcagctgat caattatacc aagagcggca
     3301 agaagttctg gaacgtgttc cacctgcagc cgatgcgcga ttataagggc gacgtccagt
     3361 acttcattgg cgtgcagctg gatggcaccg aacgtcttca tggcgccgct gagcgtgagg
     3421 cggtctgcct gatcaaaaag acagcctttc agattgctga ggcagcgaac gacgaaaatt
     3481 actttggaag cgggagtggg agcaaaaaga agaaaaagaa gtcaaagaca aagtgtgtaa
     3541 ttatgtaggc ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta
     3601 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     3661 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3721 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3781 aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     3841 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     3901 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     3961 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     4021 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4081 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     4141 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4201 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     4261 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4321 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4381 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     4441 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     4501 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4561 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4621 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4681 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4741 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4801 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4861 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4921 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     4981 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     5041 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     5101 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     5161 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     5221 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     5281 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     5341 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     5401 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     5461 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     5521 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     5581 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     5641 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5701 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     5761 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     5821 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     5881 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     5941 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     6001 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     6061 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6121 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6301 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6781 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6841 ttatcccctg attctgtgg
//
