LOCUS       sequence-341704-        6349 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of mVenus-NES-SspB-LARG-DH-P2A-iLIDcaax 
            (opto-RhoA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6349)
  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 6349)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-LARG-DH-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..6349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        55..358
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        359..562
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     513..533
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      601..610
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(607..609,610..612,613..1323)
                     /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(652..673)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(913..932)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1260..1281
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1372..1701
                     /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             2431..2487
                     /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             2509..2946
                     /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    3131..3252
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3168..3187)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3222..3241
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3259..3714)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3396..3417)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3608..3627
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3741..3845
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3847..4204
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3847..4038
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3870..3890)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4055..4190
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4239..5033
                     /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             4239..5033
                     /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(4293..4312)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4903..4922
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5221..5240)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5265..5312
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5641..6229
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6130..6149
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
       61 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      121 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      181 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      241 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      301 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      361 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      421 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      481 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      541 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggtc
      601 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      661 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      721 acctacggca agctgaccct gaagttcatc agcaccaccg gcaagctgcc cgtgccctgg
      781 cccaccctcg tgaccaccct cggctacggc ctgcagtgct tcgcccgcta ccccgaccac
      841 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      901 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
      961 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1021 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcaccgc cgacaagcag
     1081 aagaacggca tcaaggcaaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag
     1141 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1201 aaccactacc tgagctacca gtccaaactg agcaaagacc ccaacgagaa gcgcgatcac
     1261 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1321 aagttcggca ttgatctgag cggcctgacc ctgggtacca gtgctggagg tagctccccg
     1381 aaacgcccta agctgctgcg tgaatattac gattggctgg ttgataacag ctttacccca
     1441 tatctggtgg tggatgccac atacctgggc gtgaacgtgc ccgtggagta tgtgaaagac
     1501 ggtcagatcg tgctgaatct gtctgcaagt gcgaccggca acctgcaact gacaaatgat
     1561 tttatccagt tcaacgcccg ctttaagggc gtgtctcgtg aactgtatat cccgatgggt
     1621 gccgctctgg ccatttacgc tcgcgagaac ggcgatggtg tgatgttcga accagaagaa
     1681 atctatgacg agctgaatat tggtctcgac agtgctggag gtagtgctgg aggttcagct
     1741 ggtggactcg agccacccaa ctggcagcag cttgttagtc gagaagtgtt actgggacta
     1801 aaaccttgtg aaatcaaaag acaggaagtg attaatgaat tgttctacac tgaaagagct
     1861 catgttcgaa cactgaaggt tcttgatcaa gtgttctatc agcgagtatc cagagaagga
     1921 attctgtcac cctcagagct acggaaaatt ttttcaaact tggaagatat tcttcaactt
     1981 catattggat tgaatgaaca aatgaaggct gttcgaaaga gaaatgagac ctctgttatc
     2041 gatcagattg gggaagattt gctgacatgg ttcagcggac caggagagga gaaattgaaa
     2101 catgctgctg ctaccttttg cagtaaccaa cctttcgccc tggaaatgat caaatctcgt
     2161 cagaaaaagg attctcgatt tcagactttt gtgcaagatg ctgaaagtaa tccactgtgt
     2221 cgtcgtcttc aactgaagga tattattccc actcaaatgc aaaggcttac taagtaccca
     2281 cttctgttgg ataatattgc caaatacaca gaatggccaa cagaaaggga gaaggtgaag
     2341 aaagctgcag atcactgtcg tcagatctta aattatgtaa atcaggctgt caaggaggca
     2401 gaaaacaagc agcgcccgcg gggaagcgga gctactaact tcagcctgct gaagcaggct
     2461 ggagacgtgg aggagaaccc tggacctgca ccgggatccg gggagtttct ggcaaccaca
     2521 ctggaacgga tcgagaaaaa tttcgtgatt actgatccga gactgcctga caacccaatc
     2581 atttttgcga gcgattcctt cctgcagctg acagaatatt ctcgggaaga gatcctgggg
     2641 cgcaattgcc gttttctgca gggacccgag acagaccgtg ccactgttcg gaaaatcaga
     2701 gatgctattg acaaccagac tgaagtgacc gttcagctga tcaattatac caagagcggc
     2761 aagaagttct ggaacgtgtt ccacctgcag ccgatgcgcg attataaggg cgacgtccag
     2821 tacttcattg gcgtgcagct ggatggcacc gaacgtcttc atggcgccgc tgagcgtgag
     2881 gcggtctgcc tgatcaaaaa gacagccttt cagattgctg aggcagcgaa cgacgaaaat
     2941 tactttggaa gcgggagtgg gagcaaaaag aagaaaaaga agtcaaagac aaagtgtgta
     3001 attatgtagg cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
     3061 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     3121 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3181 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3241 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     3301 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3361 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3421 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3481 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3541 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3601 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3661 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     3721 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3781 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3841 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3901 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3961 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4021 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4081 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4141 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4201 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     4261 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     4321 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     4381 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     4441 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     4501 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     4561 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     4621 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4681 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4741 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     4801 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4861 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4921 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4981 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     5041 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     5101 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     5161 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     5221 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     5281 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     5341 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     5401 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     5461 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     5521 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5581 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5641 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5701 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5761 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5821 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5881 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5941 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6001 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6061 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6121 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6181 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6241 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6301 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagtta
//
