LOCUS       sequence-346237-        4670 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 4670)
  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 4670)
  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: pEntr1a-SspB-HaloTag-TIAM1-DHPH
FEATURES             Location/Qualifiers
     source          1..4670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     316..339
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             497..826
                     /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      842..851
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2899..2998)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3110..3129)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3121..3930
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3188..3207)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4020..4608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4509..4528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac
      481 tggatccggt accatgagct ccccgaaacg ccctaagctg ctgcgtgaat attacgattg
      541 gctggttgat aacagcttta ccccatatct ggtggtggat gccacatacc tgggcgtgaa
      601 cgtgcccgtg gagtatgtga aagacggtca gatcgtgctg aatctgtctg caagtgcgac
      661 cggcaacctg caactgacaa atgattttat ccagttcaac gcccgcttta agggcgtgtc
      721 tcgtgaactg tatatcccga tgggtgccgc tctggccatt tacgctcgcg agaacggcga
      781 tggtgtgatg ttcgaaccag aagaaatcta tgacgagctg aatattggta gtggaccggt
      841 cgccaccatg gccgagatcg gcaccggctt ccccttcgac ccccactacg tggaggtgct
      901 gggcgagcgc atgcactacg ttgacgtggg cccccgcgac ggcacccccg tgctgttcct
      961 gcacggcaac cccaccagca gctacgtgtg gcgcaacatc atcccccacg tggcccccac
     1021 ccaccgcact atcgcccccg acctgatcgg catgggcaag agcgacaagc ccgacctggg
     1081 ctacttcttc gacgaccacg tgcgcttcat ggacgccttc atcgaggccc tgggcctgga
     1141 ggaggtggtg ctggtgatcc acgactgggg cagcgccctg ggcttccact gggccaagcg
     1201 caaccccgag cgcgtgaagg gcatcgcctt catggagttc atccgcccca tccccacctg
     1261 ggacgagtgg cccgagttcg cccgcgagac cttccaggcc ttccgcacca ccgacgtggg
     1321 ccgcaagctg atcatcgacc agaacgtgtt catcgagggc accctgccca tgggcgtggt
     1381 gcgccccctg accgaggtgg agatggacca ctaccgcgag cccttcctga accccgtgga
     1441 ccgcgagccc ctgtggcgct tccccaacga gctgcccatc gccggcgagc ccgccaacat
     1501 cgtggccctg gtggaggagt acatggactg gctgcaccag agccccgtgc ccaagctgct
     1561 gttctggggc acccccggcg tgctgatccc ccccgccgag gccgcccgcc tggccaagag
     1621 cctgcccaac gttaaggccg tggacatcgg ccccggcctg aacctgctcc aggaggacaa
     1681 ccccgacctg atcggcagcg agatcgcccg ctggctgagc accctggaga tcagcggcgt
     1741 gtacaagtcc ggactcagat ctcgagaaca gctgtcggat gcggataagc tgcgcaaggt
     1801 gatctgtgaa ctgctggaga ctgaacgcac ctatgtgaaa gacttaaact gcctcatgga
     1861 gagatacctg aagccccttc agaaggagac ctttctcacc caggatgagc ttgatgtact
     1921 ttttggaaat ttaaccgaaa tggtggagtt tcaagtcgag ttccttaaga ctctagaaga
     1981 tggagtaaga ctggtccctg acttggaaaa gctggagaag gttgaccagt tcaagaaagt
     2041 gctcttctct ctggggggat ccttcctgta ctacgcggac cgcttcaagc tctacagtgc
     2101 cttctgtgcg agccacacaa aagtccccaa ggtcctggtg aaagccaaga cggacacagc
     2161 cttcaaggcg ttcctcgatg cccagaaccc gaggcagcag cactcatcca cgctggagtc
     2221 ttacctcatc aagcccatcc agagggtcct caagtacccg cttctgctca gggagctgtt
     2281 tgcgctgacc gatgcggaga gcgaggaaca ctaccacctg gacgtggcca tcaagactat
     2341 gaacaaggtc gccagtcaca tcaatgagat gcagaagatc cacgaagagt ttggtgccgt
     2401 gtttgaccag ctgattgctg agcagacggg agagaagaaa gaggttgcag atctgagcat
     2461 gggtgacctg ctcttgcaca ccagcgtcat ctggctgaac ccacccgcct cactgggaaa
     2521 gtggaaaaag gagccagaat tagcagcctt cgtcttcaaa acggccgtgg tccttgtata
     2581 taaagacggt tccaagcaga agaagaaact tgttggctct cacaggctgt caatctacga
     2641 ggagtgggac cccttccggt ttcgccacat gatccctacc gaagctttgc aggtccgagc
     2701 tctgcccagt gcagatgcag aggcaaatgc ggtatgcgaa attgtccacg tgaaatcaga
     2761 gtcagaaggg aggcccgagc gggttttcca cctctgctgc agttccccag agagcagaaa
     2821 ggactttctg aagtctgtgc attcgatcct gcgagataaa cacagaagac agtgagcggc
     2881 cgcactcgag atatctagac ccagctttct tgtacaaagt tggcattata agaaagcatt
     2941 gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc
     3001 atccagctgc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa
     3061 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     3121 atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat
     3181 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc
     3241 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     3301 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     3361 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     3421 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     3481 gcgctggcag tgtccctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     3541 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     3601 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     3661 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     3721 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     3781 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     3841 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     3901 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacattat
     3961 tcagattggg ccccgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4021 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4081 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4141 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     4201 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4261 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4321 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4381 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4441 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4501 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4561 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4621 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
//
