LOCUS       sequence-341712-        7426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mVenus-NES-SspB-Tiam-DHPH-P2A-iLIDcaax 
            (opto-Rac1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7426)
  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 7426)
  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-Tiam-DHPH-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..7426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             44..838
                     /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             44..838
                     /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(98..117)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     708..727
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1026..1045)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1070..1117
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1446..2034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1935..1954
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2209..2512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2513..2716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2667..2687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2755..2764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2761..2763,2764..2766,2767..3477)
                     /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(2806..2827)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3067..3086)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3414..3435
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3526..3855
                     /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             5662..5718
                     /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             5740..6177
                     /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    6362..6483
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6399..6418)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6453..6472
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(6490..6945)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6627..6648)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6839..6858
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6972..7076
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(7078..7426,1..9)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        7078..7269
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(7101..7121)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7286..7421
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
       61 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
      121 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
      181 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
      241 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
      301 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
      361 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
      421 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
      481 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
      541 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
      601 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
      661 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
      721 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
      781 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
      841 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
      901 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
      961 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     1021 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     1081 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     1141 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     1201 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     1261 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     1321 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1381 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1441 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1501 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1561 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     1621 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1681 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1741 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1801 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1861 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1921 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     1981 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2041 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2101 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta gttattaata
     2161 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     2221 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     2281 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     2341 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     2401 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     2461 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     2521 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     2581 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     2641 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     2701 ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc ggtcgccacc
     2761 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2821 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2881 ggcaagctga ccctgaagtt catcagcacc accggcaagc tgcccgtgcc ctggcccacc
     2941 ctcgtgacca ccctcggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
     3001 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     3061 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     3121 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     3181 aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac
     3241 ggcatcaagg caaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc
     3301 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     3361 tacctgagct accagtccaa actgagcaaa gaccccaacg agaagcgcga tcacatggtc
     3421 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagttc
     3481 ggcattgatc tgagcggcct gaccctgggt accagtgctg gaggtagctc cccgaaacgc
     3541 cctaagctgc tgcgtgaata ttacgattgg ctggttgata acagctttac cccatatctg
     3601 gtggtggatg ccacatacct gggcgtgaac gtgcccgtgg agtatgtgaa agacggtcag
     3661 atcgtgctga atctgtctgc aagtgcgacc ggcaacctgc aactgacaaa tgattttatc
     3721 cagttcaacg cccgctttaa gggcgtgtct cgtgaactgt atatcccgat gggtgccgct
     3781 ctggccattt acgctcgcga gaacggcgat ggtgtgatgt tcgaaccaga agaaatctat
     3841 gacgagctga atattggtct cgacagtgct ggaggtagtg ctggaggttc agctggtgga
     3901 ctcgagatga acccctctga ccagagccca tctcctcagg actccacggg gcctcagctg
     3961 gcgaccatga gacaactctc ggatgcagat aagctgcgca aggtgatctg cgagctcctg
     4021 gagacggagc gcacctacgt gaaggattta aactgtctta tggagagata cctaaagcct
     4081 cttcaaaaag aaacttttct cacccaggat gagcttgacg tgctttttgg aaatttaacg
     4141 gaaatggtag agtttcaagt agaattcctt aaaactctag aagatggagt gagactggta
     4201 cctgatttgg aaaagcttga gaaggttgat caatttaaga aagtgctgtt ctctctgggg
     4261 ggatcattcc tgtattatgc tgaccgcttc aagctctaca gtgccttctg cgccagccac
     4321 acaaaagttc ccaaggtcct ggtgaaagcc aagacagaca cggctttcaa ggcattcttg
     4381 gatgcccaga acccgaagca gcagcactca tccacgctgg agtcgtacct catcaagccc
     4441 atccagagga tcctcaagta cccacttctg ctcagggagc tgttcgccct gaccgatgcg
     4501 gagagcgagg agcactacca cctggacgtg gccatcaaga ccatgaacaa ggttgccagt
     4561 cacatcaatg agatgcagaa aatccatgaa gagtttgggg ctgtgtttga ccagctgatt
     4621 gctgaacaga ctggtgggaa aaaagaggtt gcagatctga gcatgggaga cctgcttttg
     4681 cacactaccg tgatctggct gaacccgccg gcctcgctgg gcaagtggaa aaaggaacca
     4741 gagttggcag cattcgtctt caaaactgct gtggtccttg tgtataaaga tggttccaaa
     4801 cagaagaaga aacttgtagg atctcacagg ctttccattt atgaggactg ggaccccttc
     4861 agatttcgac acatgatccc cacggaagcg ctgcaggttc gagctttggc gagtgcagat
     4921 gcagaggcaa atgccgtgtg tgaaattgtc catgtaaaat ccgagtctga agggaggccg
     4981 gagagggtct ttcacttgtg ctgcagctcc ccagagagcc gaaaggattt cctaaaggct
     5041 gtgcattcaa tcctgcgtga taagcacaga agacagctcc tcaaaaccga gagccttccc
     5101 tcatcccagc aatatgtccc ttttggaggc aaaagattgt gtgcactgaa gggggccagg
     5161 ccggccatga gcagggcagt gtctgcccca agcaagtctc ttgggaggag gaggcggcgg
     5221 ctggctcgaa acaggtttac cattgattct gatgccgtct ccgcaagcag cccggagaaa
     5281 gagtcccagc agccccccgg tggcggggac actgaccgat gggtagagga gcagtttgat
     5341 cttgctcagt atgaggagca agatgacatc aaggagacag acatcctcag tgacgatgat
     5401 gagttctgtg agtccgtgaa gggtgcctca gtggacagag acctgcagga gcggcttcag
     5461 gccacctcca tcagtcagcg ggaaagaggc cggaaaaccc tggatagtca cgcgtcccgc
     5521 atggcacagc tcaagaagca agctgccctg tcggggatca atggaggcct ggagagcgca
     5581 agcgaggaag tcatttgggt taggcgtgaa gactttgccc cctccaggaa actgaacact
     5641 gagatcccgc ggggaagcgg agctactaac ttcagcctgc tgaagcaggc tggagacgtg
     5701 gaggagaacc ctggacctgc accgggatcc ggggagtttc tggcaaccac actggaacgg
     5761 atcgagaaaa atttcgtgat tactgatccg agactgcctg acaacccaat catttttgcg
     5821 agcgattcct tcctgcagct gacagaatat tctcgggaag agatcctggg gcgcaattgc
     5881 cgttttctgc agggacccga gacagaccgt gccactgttc ggaaaatcag agatgctatt
     5941 gacaaccaga ctgaagtgac cgttcagctg atcaattata ccaagagcgg caagaagttc
     6001 tggaacgtgt tccacctgca gccgatgcgc gattataagg gcgacgtcca gtacttcatt
     6061 ggcgtgcagc tggatggcac cgaacgtctt catggcgccg ctgagcgtga ggcggtctgc
     6121 ctgatcaaaa agacagcctt tcagattgct gaggcagcga acgacgaaaa ttactttgga
     6181 agcgggagtg ggagcaaaaa gaagaaaaag aagtcaaaga caaagtgtgt aattatgtag
     6241 gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     6301 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     6361 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     6421 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     6481 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     6541 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     6601 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     6661 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     6721 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     6781 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     6841 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     6901 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     6961 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     7021 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     7081 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     7141 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     7201 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     7261 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     7321 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     7381 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggc
//
