LOCUS       sequence-341714-        6739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mVenus-NES-SspB-ITSN1-DHPH-P2A-iLIDcaax 
            (opto-Cdc42).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6739)
  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 6739)
  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-ITSN1-DHPH-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        184..487
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        488..691
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     642..662
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      730..739
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(736..738,739..741,742..1452)
                     /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(781..802)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1042..1061)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1389..1410
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1501..1830
                     /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             2950..3006
                     /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             3028..3465
                     /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    3650..3771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3687..3706)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3741..3760
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3778..4233)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3915..3936)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4127..4146
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4260..4364
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4366..4723
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4366..4557
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4389..4409)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4574..4709
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4758..5552
                     /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             4758..5552
                     /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(4812..4831)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5422..5441
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5740..5759)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5784..5831
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      join(6160..6739,1..9)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6649..6668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
       61 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
      121 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      181 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      241 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      301 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      361 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      421 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      481 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      541 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      601 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      661 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg
      721 ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      781 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
      841 ggcgatgcca cctacggcaa gctgaccctg aagttcatca gcaccaccgg caagctgccc
      901 gtgccctggc ccaccctcgt gaccaccctc ggctacggcc tgcagtgctt cgcccgctac
      961 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1021 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1081 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1141 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcaccgcc
     1201 gacaagcaga agaacggcat caaggcaaac ttcaagatcc gccacaacat cgaggacggc
     1261 ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1321 ctgcccgaca accactacct gagctaccag tccaaactga gcaaagaccc caacgagaag
     1381 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1441 gagctgtaca agttcggcat tgatctgagc ggcctgaccc tgggtaccag tgctggaggt
     1501 agctccccga aacgccctaa gctgctgcgt gaatattacg attggctggt tgataacagc
     1561 tttaccccat atctggtggt ggatgccaca tacctgggcg tgaacgtgcc cgtggagtat
     1621 gtgaaagacg gtcagatcgt gctgaatctg tctgcaagtg cgaccggcaa cctgcaactg
     1681 acaaatgatt ttatccagtt caacgcccgc tttaagggcg tgtctcgtga actgtatatc
     1741 ccgatgggtg ccgctctggc catttacgct cgcgagaacg gcgatggtgt gatgttcgaa
     1801 ccagaagaaa tctatgacga gctgaatatt ggtctcgaca gtgctggagg tagtgctgga
     1861 ggttcagctg gtggactcga gatgttgacc ccaactgaaa gaaagcgaca aggatacatc
     1921 cacgagctca ttgtcaccga ggagaactat gtgaatgacc tgcagctggt cacagagatt
     1981 tttcaaaaac ccctgatgga gtctgagctg ctgacagaaa aagaggttgc tatgattttt
     2041 gtgaactgga aggagctgat tatgtgtaat atcaaactac taaaagcgct gagagtccgc
     2101 aagaagatgt ccggggagaa gatgcctgtg aagatgattg gagacatcct gagcgcacag
     2161 ctgccgcaca tgcagcccta catccgcttc tgcagccgcc agctcaacgg ggctgccctg
     2221 atccagcaga agacggatga ggccccagac ttcaaggagt tcgtcaaaag attggcaatg
     2281 gatcctcggt gtaaagggat gccactctct agttttatac tgaagcctat gcaacgggta
     2341 acaagatacc cactgatcat taaaaatatc ctggaaaaca cccctgaaaa ccacccggac
     2401 cacagccact tgaagcacgc cctggagaag gcggaagagc tctgttccca ggtgaacgaa
     2461 ggggtgcggg agaaggagaa ctctgaccgg ctggagtgga tccaggccca cgtgcagtgt
     2521 gaaggcctgt ctgagcaact tgtgttcaat tcagtgacca attgcttggg gccgcgcaaa
     2581 tttctgcaca gtgggaagct ctacaaggcc aagagcaaca aggagctgta tggcttcctt
     2641 ttcaacgact tcctcctgct gactcagatc acgaagcctt tggggtcttc tggcaccgac
     2701 aaagtcttca gccccaaatc aaacctgcag tataaaatgt ataaaacacc tattttccta
     2761 aatgaggttc tagtaaaatt acccaccgac ccttctggag acgagcccat cttccacatc
     2821 tcccacattg accgcgtcta tactctccga gcagaaagca taaatgaaag gactgcctgg
     2881 gtgcagaaaa tcaaagctgc ttctgaactc tacatagaga ctgagaaaaa gaagccgcgg
     2941 ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct
     3001 ggacctgcac cgggatccgg ggagtttctg gcaaccacac tggaacggat cgagaaaaat
     3061 ttcgtgatta ctgatccgag actgcctgac aacccaatca tttttgcgag cgattccttc
     3121 ctgcagctga cagaatattc tcgggaagag atcctggggc gcaattgccg ttttctgcag
     3181 ggacccgaga cagaccgtgc cactgttcgg aaaatcagag atgctattga caaccagact
     3241 gaagtgaccg ttcagctgat caattatacc aagagcggca agaagttctg gaacgtgttc
     3301 cacctgcagc cgatgcgcga ttataagggc gacgtccagt acttcattgg cgtgcagctg
     3361 gatggcaccg aacgtcttca tggcgccgct gagcgtgagg cggtctgcct gatcaaaaag
     3421 acagcctttc agattgctga ggcagcgaac gacgaaaatt actttggaag cgggagtggg
     3481 agcaaaaaga agaaaaagaa gtcaaagaca aagtgtgtaa ttatgtaggc ggccgcgact
     3541 ctagatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     3601 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     3661 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3721 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa
     3781 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     3841 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     3901 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     3961 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     4021 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4081 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     4141 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     4201 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     4261 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4321 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa
     4381 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     4441 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4501 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4561 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4621 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4681 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     4741 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4801 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4861 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4921 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4981 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     5041 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     5101 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     5161 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     5221 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     5281 aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     5341 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     5401 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     5461 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     5521 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     5581 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     5641 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     5701 catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat
     5761 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg
     5821 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc
     5881 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag
     5941 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc
     6001 aggttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6061 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6121 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6181 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6241 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6301 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6361 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6421 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6481 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6541 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6601 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6661 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6721 ctcgtcaggg gggcggagc
//
