LOCUS       sequence-346370-        6381 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 6381)
  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 6381)
  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-iRFP-DHPH-TIAM1
FEATURES             Location/Qualifiers
     source          1..6381
                     /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..1887
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     polyA_signal    3169..3290
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3206..3225)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3260..3279
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3297..3752)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3434..3455)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3646..3665
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3779..3883
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3885..4242
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3885..4076
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3908..3928)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4093..4228
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4277..5071
                     /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             4277..5071
                     /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(4331..4350)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4941..4960
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5259..5278)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5303..5350
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5679..6267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6168..6187
                     /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 caccatggcg
      961 cgtaaggtcg atctcacctc ctgcgatcgc gagccgatcc acatccccgg cagcattcag
     1021 ccgtgcggct gcctgctagc ctgcgacgcg caggcggtgc ggatcacgcg cattacggaa
     1081 aatgccggcg cgttctttgg acgcgaaact ccgcgggtcg gtgagctact cgccgattac
     1141 ttcggcgaga ccgaagccca tgcgctgcgc aacgcactgg cgcagtcctc cgatccaaag
     1201 cgaccggcgc tgatcttcgg ttggcgcgac ggcctgaccg gccgcacctt cgacatctca
     1261 ctgcatcgcc atgacggtac atcgatcatc gagttcgagc ctgcggcggc cgaacaggcc
     1321 gacaatccgc tgcggctgac gcggcagatc atcgcgcgca ccaaagaact gaagtcgctc
     1381 gaagagatgg ccgcacgggt gccgcgctat ctgcaggcga tgctcggcta tcaccgcgtg
     1441 atgttgtacc gcttcgcgga cgacggctcc gggatggtga tcggcgaggc gaagcgcagc
     1501 gacctcgaga gctttctcgg tcagcacttt ccggcgtcgc tggtcccgca gcaggcgcgg
     1561 ctactgtact tgaagaacgc gatccgcgtg gtctcggatt cgcgcggcat cagcagccgg
     1621 atcgtgcccg agcacgacgc ctccggcgcc gcgctcgatc tgtcgttcgc gcacctgcgc
     1681 agcatctcgc cctgccatct cgaatttctg cggaacatgg gcgtcagcgc ctcgatgtcg
     1741 ctgtcgatca tcattgacgg cacgctatgg ggattgatca tctgtcatca ttacgagccg
     1801 cgtgccgtgc cgatggcgca gcgcgtcgcg gccgaaatgt tcgccgactt cttatcgctg
     1861 cacttcaccg ccgcccacca ccaacgcgtg tacaagtccg gactcagatc tcgagaacag
     1921 ctgtcggatg cggataagct gcgcaaggtg atctgtgaac tgctggagac tgaacgcacc
     1981 tatgtgaaag acttaaactg cctcatggag agatacctga agccccttca gaaggagacc
     2041 tttctcaccc aggatgagct tgatgtactt tttggaaatt taaccgaaat ggtggagttt
     2101 caagtcgagt tccttaagac tctagaagat ggagtaagac tggtccctga cttggaaaag
     2161 ctggagaagg ttgaccagtt caagaaagtg ctcttctctc tggggggatc cttcctgtac
     2221 tacgcggacc gcttcaagct ctacagtgcc ttctgtgcga gccacacaaa agtccccaag
     2281 gtcctggtga aagccaagac ggacacagcc ttcaaggcgt tcctcgatgc ccagaacccg
     2341 aggcagcagc actcatccac gctggagtct tacctcatca agcccatcca gagggtcctc
     2401 aagtacccgc ttctgctcag ggagctgttt gcgctgaccg atgcggagag cgaggaacac
     2461 taccacctgg acgtggccat caagactatg aacaaggtcg ccagtcacat caatgagatg
     2521 cagaagatcc acgaagagtt tggtgccgtg tttgaccagc tgattgctga gcagacggga
     2581 gagaagaaag aggttgcaga tctgagcatg ggtgacctgc tcttgcacac cagcgtcatc
     2641 tggctgaacc cacccgcctc actgggaaag tggaaaaagg agccagaatt agcagccttc
     2701 gtcttcaaaa cggccgtggt ccttgtatat aaagacggtt ccaagcagaa gaagaaactt
     2761 gttggctctc acaggctgtc aatctacgag gagtgggacc ccttccggtt tcgccacatg
     2821 atccctaccg aagctttgca ggtccgagct ctgcccagtg cagatgcaga ggcaaatgcg
     2881 gtatgcgaaa ttgtccacgt gaaatcagag tcagaaggga ggcccgagcg ggttttccac
     2941 ctctgctgca gttccccaga gagcagaaag gactttctga agtctgtgca ttcgatcctg
     3001 cgagataaac acagaagaca gtaaggtacc gcgggcccgg gatccaccgg atctagataa
     3061 ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     3121 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     3181 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3241 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat
     3301 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     3361 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     3421 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     3481 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     3541 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     3601 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     3661 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     3721 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     3781 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3841 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     3901 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3961 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4021 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4081 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4141 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4201 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     4261 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4321 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4381 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4441 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4501 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4561 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4621 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4681 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4741 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4801 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4861 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4921 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4981 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5041 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5101 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5161 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5221 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     5281 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     5341 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     5401 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     5461 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     5521 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5581 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5641 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5701 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5761 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5821 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5881 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5941 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6001 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6061 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6121 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6181 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6241 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6301 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6361 aaccgtatta ccgccatgca t
//
