LOCUS       sequence-346249-        4558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterodimerization with FRB (rapamycin), Rac1 activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4558)
  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 4558)
  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-mcherry-FKBP-TIAM1(DHPH)
FEATURES             Location/Qualifiers
     source          1..4558
                     /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             481..1188
                     /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(626..644)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     894..913
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1201..1521
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     protein_bind    complement(2787..2886)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2998..3017)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3009..3818
                     /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(3076..3095)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3908..4496
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4397..4416
                     /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 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
      541 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
      601 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
      661 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
      721 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
      781 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
      841 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
      901 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
      961 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     1021 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     1081 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     1141 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagag tgctggtggt
     1201 ggagtgcagg tggaaaccat ctccccagga gacgggcgca ccttccccaa gcgcggccag
     1261 acctgcgtgg tgcactacac cgggatgctt gaagatggaa agaaatttga ttcctcccgg
     1321 gacagaaaca agccctttaa gtttatgcta ggcaagcagg aggtgatccg aggctgggaa
     1381 gaaggggttg cccagatgag tgtgggtcag agagccaaac tgactatatc tccagattat
     1441 gcctatggtg ccactgggca cccaggcatc atcccaccac atgccactct cgtcttcgat
     1501 gtggagcttc taaaactgga aagtgctggt ggtagtgctg gtggtagtgc tggtggtagt
     1561 gctggtggta gtgctggtgg tcctcgagca atgaacccct ctgaccagag cccatctcct
     1621 caggactcca cggggcctca gctggcgacc atgagacaac tctcggatgc agataagctg
     1681 cgcaaggtga tctgcgagct cctggagacg gagcgcacct acgtgaagga tttaaactgt
     1741 cttatggaga gatacctaaa gcctcttcaa aaagaaactt ttctcaccca ggatgagctt
     1801 gacgtgcttt ttggaaattt aacggaaatg gtagagtttc aagtagaatt ccttaaaact
     1861 ctagaagatg gagtgagact ggtacctgat ttggaaaagc ttgagaaggt tgatcaattt
     1921 aagaaagtgc tgttctctct ggggggatca ttcctgtatt atgctgaccg cttcaagctc
     1981 tacagtgcct tctgcgccag ccacacaaaa gttcccaagg tcctggtgaa agccaagaca
     2041 gacacggctt tcaaggcatt cttggatgcc cagaacccga agcagcagca ctcatccacg
     2101 ctggagtcgt acctcatcaa gcccatccag aggatcctca agtacccact tctgctcagg
     2161 gagctgttcg ccctgaccga tgcggagagc gaggagcact accacctgga cgtggccatc
     2221 aagaccatga acaaggttgc cagtcacatc aatgagatgc agaaaatcca tgaagagttt
     2281 ggggctgtgt ttgaccagct gattgctgaa cagactggtg ggaaaaaaga ggttgcagat
     2341 ctgagcatgg gagacctgct tttgcacact accgtgatct ggctgaaccc gccggcctcg
     2401 ctgggcaagt ggaaaaagga accagagttg gcagcattcg tcttcaaaac tgctgtggtc
     2461 cttgtgtata aagatggttc caaacagaag aagaaacttg taggatctca caggctttcc
     2521 atttatgagg actgggaccc cttcagattt cgacacatga tccccacgga agcgctgcag
     2581 gttcgagctt tggcgagtgc agatgcagag gcaaatgccg tgtgtgaaat tgtccatgta
     2641 aaatccgagt ctgaagggag gccggagagg gtctttcact tgtgctgcag ctccccagag
     2701 agccgaaagg atttcctaaa ggctgtgcat tcaatcctgc gtgataagca cagaagacag
     2761 tgagcggccg cactcgagat atctagaccc agctttcttg tacaaagttg gcattataag
     2821 aaagcattgc ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat
     2881 tatttgccat ccagctgcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac
     2941 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     3001 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
     3061 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     3121 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     3181 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     3241 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     3301 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     3361 ttgttgatgc gctggcagtg tccctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     3421 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     3481 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     3541 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     3601 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     3661 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     3721 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     3781 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     3841 aacattattc agattgggcc ccgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3901 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3961 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4021 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4081 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4141 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4201 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4261 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4321 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4381 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4441 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4501 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtt
//
