LOCUS       sequence-346371-        6339 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 6339)
  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 6339)
  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-HaloTag-DHPH-TIAM1
FEATURES             Location/Qualifiers
     source          1..6339
                     /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"
     polyA_signal    3127..3248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3164..3183)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3218..3237
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3255..3710)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3392..3413)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3604..3623
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3737..3841
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3843..4200
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3843..4034
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3866..3886)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4051..4186
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4235..5029
                     /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             4235..5029
                     /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(4289..4308)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4899..4918
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5217..5236)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5261..5308
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5637..6225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6126..6145
                     /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 caccatggcc
      961 gagatcggca ccggcttccc cttcgacccc cactacgtgg aggtgctggg cgagcgcatg
     1021 cactacgttg acgtgggccc ccgcgacggc acccccgtgc tgttcctgca cggcaacccc
     1081 accagcagct acgtgtggcg caacatcatc ccccacgtgg cccccaccca ccgcactatc
     1141 gcccccgacc tgatcggcat gggcaagagc gacaagcccg acctgggcta cttcttcgac
     1201 gaccacgtgc gcttcatgga cgccttcatc gaggccctgg gcctggagga ggtggtgctg
     1261 gtgatccacg actggggcag cgccctgggc ttccactggg ccaagcgcaa ccccgagcgc
     1321 gtgaagggca tcgccttcat ggagttcatc cgccccatcc ccacctggga cgagtggccc
     1381 gagttcgccc gcgagacctt ccaggccttc cgcaccaccg acgtgggccg caagctgatc
     1441 atcgaccaga acgtgttcat cgagggcacc ctgcccatgg gcgtggtgcg ccccctgacc
     1501 gaggtggaga tggaccacta ccgcgagccc ttcctgaacc ccgtggaccg cgagcccctg
     1561 tggcgcttcc ccaacgagct gcccatcgcc ggcgagcccg ccaacatcgt ggccctggtg
     1621 gaggagtaca tggactggct gcaccagagc cccgtgccca agctgctgtt ctggggcacc
     1681 cccggcgtgc tgatcccccc cgccgaggcc gcccgcctgg ccaagagcct gcccaacgtt
     1741 aaggccgtgg acatcggccc cggcctgaac ctgctccagg aggacaaccc cgacctgatc
     1801 ggcagcgaga tcgcccgctg gctgagcacc ctggagatca gcggcgtgta caagtccgga
     1861 ctcagatctc gagaacagct gtcggatgcg gataagctgc gcaaggtgat ctgtgaactg
     1921 ctggagactg aacgcaccta tgtgaaagac ttaaactgcc tcatggagag atacctgaag
     1981 ccccttcaga aggagacctt tctcacccag gatgagcttg atgtactttt tggaaattta
     2041 accgaaatgg tggagtttca agtcgagttc cttaagactc tagaagatgg agtaagactg
     2101 gtccctgact tggaaaagct ggagaaggtt gaccagttca agaaagtgct cttctctctg
     2161 gggggatcct tcctgtacta cgcggaccgc ttcaagctct acagtgcctt ctgtgcgagc
     2221 cacacaaaag tccccaaggt cctggtgaaa gccaagacgg acacagcctt caaggcgttc
     2281 ctcgatgccc agaacccgag gcagcagcac tcatccacgc tggagtctta cctcatcaag
     2341 cccatccaga gggtcctcaa gtacccgctt ctgctcaggg agctgtttgc gctgaccgat
     2401 gcggagagcg aggaacacta ccacctggac gtggccatca agactatgaa caaggtcgcc
     2461 agtcacatca atgagatgca gaagatccac gaagagtttg gtgccgtgtt tgaccagctg
     2521 attgctgagc agacgggaga gaagaaagag gttgcagatc tgagcatggg tgacctgctc
     2581 ttgcacacca gcgtcatctg gctgaaccca cccgcctcac tgggaaagtg gaaaaaggag
     2641 ccagaattag cagccttcgt cttcaaaacg gccgtggtcc ttgtatataa agacggttcc
     2701 aagcagaaga agaaacttgt tggctctcac aggctgtcaa tctacgagga gtgggacccc
     2761 ttccggtttc gccacatgat ccctaccgaa gctttgcagg tccgagctct gcccagtgca
     2821 gatgcagagg caaatgcggt atgcgaaatt gtccacgtga aatcagagtc agaagggagg
     2881 cccgagcggg ttttccacct ctgctgcagt tccccagaga gcagaaagga ctttctgaag
     2941 tctgtgcatt cgatcctgcg agataaacac agaagacagt aaggtaccgc gggcccggga
     3001 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     3061 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3121 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3181 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3241 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     3301 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     3361 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     3421 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     3481 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     3541 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3601 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3661 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     3721 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3781 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3841 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3901 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3961 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4021 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4081 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4141 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4201 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     4261 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     4321 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     4381 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     4441 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4501 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4561 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4621 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4681 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4741 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4801 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4861 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4921 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4981 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     5041 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     5101 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5161 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     5221 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     5281 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     5341 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     5401 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     5461 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     5521 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5581 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5641 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5701 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5761 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5821 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5881 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5941 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6001 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6061 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6121 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6181 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6241 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6301 tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
