LOCUS       sequence-341715-        7144 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mVenus-NES-SspB-SOS2cat-P2A-iLIDcaax 
            (opto-Ras).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7144)
  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 7144)
  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-SOS2cat-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..7144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             37..831
                     /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             37..831
                     /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(91..110)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     701..720
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1019..1038)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1063..1110
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1439..2027
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1928..1947
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2202..2505
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2506..2709
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2660..2680
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2748..2757
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2754..2756,2757..2759,2760..3470)
                     /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(2799..2820)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3060..3079)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3407..3428
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3519..3848
                     /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             5373..5429
                     /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             5451..5888
                     /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    6073..6194
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6110..6129)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6164..6183
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(6201..6656)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6338..6359)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6550..6569
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6683..6787
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6789..7144,1..2)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6789..6980
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6812..6832)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6997..7132
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
       61 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
      121 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
      181 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
      241 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
      301 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
      361 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
      421 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
      481 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
      541 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
      601 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
      661 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
      721 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
      781 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
      841 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
      901 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
      961 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     1021 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     1081 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     1141 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     1201 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     1261 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     1321 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     1381 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1441 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     1501 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1561 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     1621 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     1681 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     1741 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1801 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1861 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1921 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1981 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2041 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2101 tatcccctga ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca
     2161 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     2221 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     2281 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     2341 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     2401 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     2461 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     2521 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     2581 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     2641 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     2701 agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc accatggtga
     2761 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     2821 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     2881 tgaccctgaa gttcatcagc accaccggca agctgcccgt gccctggccc accctcgtga
     2941 ccaccctcgg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     3001 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     3061 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     3121 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     3181 agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag aacggcatca
     3241 aggcaaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc gccgaccact
     3301 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     3361 gctaccagtc caaactgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     3421 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ttcggcattg
     3481 atctgagcgg cctgaccctg ggtaccagtg ctggaggtag ctccccgaaa cgccctaagc
     3541 tgctgcgtga atattacgat tggctggttg ataacagctt taccccatat ctggtggtgg
     3601 atgccacata cctgggcgtg aacgtgcccg tggagtatgt gaaagacggt cagatcgtgc
     3661 tgaatctgtc tgcaagtgcg accggcaacc tgcaactgac aaatgatttt atccagttca
     3721 acgcccgctt taagggcgtg tctcgtgaac tgtatatccc gatgggtgcc gctctggcca
     3781 tttacgctcg cgagaacggc gatggtgtga tgttcgaacc agaagaaatc tatgacgagc
     3841 tgaatattgg tctcgacagt gctggaggta gtgctggagg ttcagctggt ggactcgagc
     3901 aaccactgag attaccaagt cctgaagtat atcgttttgt agtaaaagac tctgaggaaa
     3961 acattgtttt tgaagacaac ttgcaaagta gaagtggcat ccccattatt aaaggaggaa
     4021 ctgtagtgaa attaattgaa aggttaacat atcatatgta tgcagatccc aattttgttc
     4081 gtacttttct taccacatat cgttcatttt gtaaaccaca ggaattgctg agcttactga
     4141 ttgaacggtt tgaaattcca gagccagaac ctactgacgc agacaaattg gcaatagaga
     4201 aaggcgagca gccaatcagt gcagacctta aaagatttcg caaggaatat gtccaaccag
     4261 tacaacttag gatcttaaat gtatttcggc attgggttga acatcatttt tatgactttg
     4321 aaagagactt ggaattgctt gaaagactag aatccttcat ttcaagtgta agagggaaag
     4381 ctatgaaaaa atgggtagag tcaattgcta agatcatcag gaggaagaag caagctcagg
     4441 caaacggagt aagccataat attacctttg aaagtccacc tccaccaatt gaatggcata
     4501 tcagcaaacc aggacagttt gaaacatttg atctcatgac acttcatcca atagaaattg
     4561 cacgtcagct gacacttttg gagtctgatc tttacaggaa agttcaaccg tctgaacttg
     4621 tagggagtgt gtggaccaaa gaagataaag aaataaattc tccaaattta ttaaaaatga
     4681 ttcgccatac cacaaatctc accctctggt ttgaaaaatg cattgtggaa gcagaaaatt
     4741 ttgaagaacg ggtggcagta ctaagtagaa ttatagaaat tctgcaagtt tttcaagatt
     4801 tgaataattt caatggcgta ttggagatag tcagtgcagt aaattcagtg tcagtataca
     4861 gactagacca tacctttgag gcactgcagg aaaggaaaag gaaaattttg gacgaagctg
     4921 tggaattaag tcaagatcac tttaaaaaat acctagtaaa acttaagtca atcaatccac
     4981 cttgtgtgcc tttttttgga atatatttaa caaatattct gaagaccgaa gaagggaata
     5041 atgatttttt aaaaaagaaa gggaaagatt taatcaattt cagtaagagg aggaaagtag
     5101 ctgaaattac tggagaaatt cagcagtatc agaatcagcc ttactgttta cggatagaac
     5161 cagatatgag gagattcttt gaaaacctta accccatggg aagtgcatct gaaaaagagt
     5221 ttacagatta tttgttcaac aagtcactag aaattgaacc tcgaaactgc aaacagccac
     5281 ctcgatttcc taggaaatca actttttcct taaaatctcc tggaataagg cctaacacag
     5341 gccgacatgg ctctaccccg cggggaagcg gagctactaa cttcagcctg ctgaagcagg
     5401 ctggagacgt ggaggagaac cctggacctg caccgggatc cggggagttt ctggcaacca
     5461 cactggaacg gatcgagaaa aatttcgtga ttactgatcc gagactgcct gacaacccaa
     5521 tcatttttgc gagcgattcc ttcctgcagc tgacagaata ttctcgggaa gagatcctgg
     5581 ggcgcaattg ccgttttctg cagggacccg agacagaccg tgccactgtt cggaaaatca
     5641 gagatgctat tgacaaccag actgaagtga ccgttcagct gatcaattat accaagagcg
     5701 gcaagaagtt ctggaacgtg ttccacctgc agccgatgcg cgattataag ggcgacgtcc
     5761 agtacttcat tggcgtgcag ctggatggca ccgaacgtct tcatggcgcc gctgagcgtg
     5821 aggcggtctg cctgatcaaa aagacagcct ttcagattgc tgaggcagcg aacgacgaaa
     5881 attactttgg aagcgggagt gggagcaaaa agaagaaaaa gaagtcaaag acaaagtgtg
     5941 taattatgta ggcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     6001 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     6061 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     6121 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6181 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     6241 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     6301 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     6361 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     6421 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     6481 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     6541 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     6601 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     6661 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     6721 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     6781 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     6841 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     6901 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     6961 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     7021 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     7081 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     7141 tgca
//
