LOCUS       sequence-346365-        6147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterodimerization with iLID, Rac1 activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6147)
  AUTHORS   Mahlandt EK, Palacios Martinez S, Arts JJG, Tol S, van Buul JD, 
            Goedhart J
  TITLE     Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control
            Rho GTPase activity on a global to subcellular scale, enabling 
            precise control over vascular endothelial barrier strength.
  JOURNAL   Elife. 2023 Jul 14;12:RP84364. doi: 10.7554/eLife.84364.
  PUBMED    37449837
REFERENCE   2  (bases 1 to 6147)
  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: "Elife."; 
            date: "2023-07-14"; volume: "12:RP84364. doi"; pages: " 
            10.7554/eLife.84364"
COMMENT     Sequence Label: SspB-mCherry-DHPH-TIAM1
FEATURES             Location/Qualifiers
     source          1..6147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             604..933
                     /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"
     regulatory      949..958
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             955..1662
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1100..1118)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1368..1387
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2935..3056
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2972..2991)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3026..3045
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3063..3518)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3200..3221)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3412..3431
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3545..3649
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3651..4008
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3651..3842
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3674..3694)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3859..3994
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4043..4837
                     /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             4043..4837
                     /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(4097..4116)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4707..4726
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5025..5044)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5069..5116
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5445..6033
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5934..5953
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagccacc
      601 atgagctccc cgaaacgccc taagctgctg cgtgaatatt acgattggct ggttgataac
      661 agctttaccc catatctggt ggtggatgcc acatacctgg gcgtgaacgt gcccgtggag
      721 tatgtgaaag acggtcagat cgtgctgaat ctgtctgcaa gtgcgaccgg caacctgcaa
      781 ctgacaaatg attttatcca gttcaacgcc cgctttaagg gcgtgtctcg tgaactgtat
      841 atcccgatgg gtgccgctct ggccatttac gctcgcgaga acggcgatgg tgtgatgttc
      901 gaaccagaag aaatctatga cgagctgaat attggtagtg gaccggtcgc caccatggtg
      961 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     1021 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     1081 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     1141 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     1201 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     1261 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     1321 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     1381 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     1441 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     1501 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     1561 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     1621 gagggccgcc actccaccgg cggcatggac gagctgtaca agtccggact cagatctcga
     1681 gaacagctgt cggatgcgga taagctgcgc aaggtgatct gtgaactgct ggagactgaa
     1741 cgcacctatg tgaaagactt aaactgcctc atggagagat acctgaagcc ccttcagaag
     1801 gagacctttc tcacccagga tgagcttgat gtactttttg gaaatttaac cgaaatggtg
     1861 gagtttcaag tcgagttcct taagactcta gaagatggag taagactggt ccctgacttg
     1921 gaaaagctgg agaaggttga ccagttcaag aaagtgctct tctctctggg gggatccttc
     1981 ctgtactacg cggaccgctt caagctctac agtgccttct gtgcgagcca cacaaaagtc
     2041 cccaaggtcc tggtgaaagc caagacggac acagccttca aggcgttcct cgatgcccag
     2101 aacccgaggc agcagcactc atccacgctg gagtcttacc tcatcaagcc catccagagg
     2161 gtcctcaagt acccgcttct gctcagggag ctgtttgcgc tgaccgatgc ggagagcgag
     2221 gaacactacc acctggacgt ggccatcaag actatgaaca aggtcgccag tcacatcaat
     2281 gagatgcaga agatccacga agagtttggt gccgtgtttg accagctgat tgctgagcag
     2341 acgggagaga agaaagaggt tgcagatctg agcatgggtg acctgctctt gcacaccagc
     2401 gtcatctggc tgaacccacc cgcctcactg ggaaagtgga aaaaggagcc agaattagca
     2461 gccttcgtct tcaaaacggc cgtggtcctt gtatataaag acggttccaa gcagaagaag
     2521 aaacttgttg gctctcacag gctgtcaatc tacgaggagt gggacccctt ccggtttcgc
     2581 cacatgatcc ctaccgaagc tttgcaggtc cgagctctgc ccagtgcaga tgcagaggca
     2641 aatgcggtat gcgaaattgt ccacgtgaaa tcagagtcag aagggaggcc cgagcgggtt
     2701 ttccacctct gctgcagttc cccagagagc agaaaggact ttctgaagtc tgtgcattcg
     2761 atcctgcgag ataaacacag aagacagtaa ggtaccgcgg gcccgggatc caccggatct
     2821 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     2881 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     2941 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3001 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     3061 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3121 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3181 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3241 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3301 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3361 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3421 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3481 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     3541 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3601 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     3661 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3721 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3781 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3841 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3901 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3961 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4021 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4081 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4141 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4201 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4261 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4321 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4381 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4441 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4501 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4561 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4621 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4681 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4741 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4801 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4861 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4921 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4981 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5041 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5101 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     5161 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     5221 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     5281 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5341 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5401 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5461 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5521 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5581 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5641 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5701 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5761 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5821 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5881 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5941 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6001 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6061 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6121 gtggataacc gtattaccgc catgcat
//
