LOCUS       sequence-341705-        5710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mVenus-NES-SspB-P2A-iLIDcaax (opto-control) 
            containing multiple cloning sites (MCS) .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5710)
  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 5710)
  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-MCS-P2A-iLIDcaax
FEATURES             Location/Qualifiers
     source          1..5710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        151..454
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        455..658
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     609..629
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      697..706
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(703..705,706..708,709..1419)
                     /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(748..769)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1009..1028)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1356..1377
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1468..1797
                     /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             1888..1944
                     /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             1966..2403
                     /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    2588..2709
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2625..2644)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2679..2698
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2716..3171)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2853..2874)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3065..3084
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3198..3302
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3304..3661
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3304..3495
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3327..3347)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3512..3647
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3696..4490
                     /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             3696..4490
                     /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(3750..3769)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4360..4379
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4678..4697)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4722..4769
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5098..5686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5587..5606
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
       61 tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc
      121 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      181 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      361 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      421 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      481 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      541 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      601 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
      661 tttagtgaac cgtcagatcc gctagcgcta ccggtcgcca ccatggtgag caagggcgag
      721 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      781 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      841 ttcatcagca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctcggc
      901 tacggcctgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
      961 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     1021 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1081 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1141 aacagccaca acgtctatat caccgccgac aagcagaaga acggcatcaa ggcaaacttc
     1201 aagatccgcc acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac
     1261 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc
     1321 aaactgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1381 gccgccggga tcactctcgg catggacgag ctgtacaagt tcggcattga tctgagcggc
     1441 ctgaccctgg gtaccagtgc tggaggtagc tccccgaaac gccctaagct gctgcgtgaa
     1501 tattacgatt ggctggttga taacagcttt accccatatc tggtggtgga tgccacatac
     1561 ctgggcgtga acgtgcccgt ggagtatgtg aaagacggtc agatcgtgct gaatctgtct
     1621 gcaagtgcga ccggcaacct gcaactgaca aatgatttta tccagttcaa cgcccgcttt
     1681 aagggcgtgt ctcgtgaact gtatatcccg atgggtgccg ctctggccat ttacgctcgc
     1741 gagaacggcg atggtgtgat gttcgaacca gaagaaatct atgacgagct gaatattggt
     1801 ctcgacagtg ctggaggtag tgctggaggt tcagctggtg gactcgaggt cgacgaattc
     1861 gagctcgata tcccgcgggg aagcggagct actaacttca gcctgctgaa gcaggctgga
     1921 gacgtggagg agaaccctgg acctgcaccg ggatccgggg agtttctggc aaccacactg
     1981 gaacggatcg agaaaaattt cgtgattact gatccgagac tgcctgacaa cccaatcatt
     2041 tttgcgagcg attccttcct gcagctgaca gaatattctc gggaagagat cctggggcgc
     2101 aattgccgtt ttctgcaggg acccgagaca gaccgtgcca ctgttcggaa aatcagagat
     2161 gctattgaca accagactga agtgaccgtt cagctgatca attataccaa gagcggcaag
     2221 aagttctgga acgtgttcca cctgcagccg atgcgcgatt ataagggcga cgtccagtac
     2281 ttcattggcg tgcagctgga tggcaccgaa cgtcttcatg gcgccgctga gcgtgaggcg
     2341 gtctgcctga tcaaaaagac agcctttcag attgctgagg cagcgaacga cgaaaattac
     2401 tttggaagcg ggagtgggag caaaaagaag aaaaagaagt caaagacaaa gtgtgtaatt
     2461 atgtaggcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     2521 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2581 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2641 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2701 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     2761 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2821 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2881 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2941 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     3001 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     3061 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     3121 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     3181 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3241 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3301 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3361 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3421 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3481 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3541 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3601 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3661 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3721 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3781 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3841 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     3901 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3961 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     4021 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4081 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     4141 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4201 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     4261 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4321 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4381 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4441 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4501 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4561 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4621 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
     4681 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     4741 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
     4801 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
     4861 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
     4921 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt
     4981 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5041 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5101 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5161 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5221 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5281 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5341 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5401 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5461 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5521 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5581 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5641 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5701 ggccttttta
//
