LOCUS       sequence-346328-        6343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterodimerization with iLID, Cdc42 activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6343)
  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 6343)
  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-ITSN1
FEATURES             Location/Qualifiers
     source          1..6343
                     /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    3131..3252
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3168..3187)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3222..3241
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3259..3714)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3396..3417)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3608..3627
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3741..3845
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3847..4204
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3847..4038
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3870..3890)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4055..4190
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4239..5033
                     /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             4239..5033
                     /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(4293..4312)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4903..4922
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5221..5240)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5265..5312
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5641..6229
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6130..6149
                     /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 tgatatgttg
     1921 accccaactg aaagaaagcg acaaggatac atccacgagc tcattgtcac cgaggagaac
     1981 tatgtgaatg acctgcagct ggtcacagag atttttcaaa aacccctgat ggagtctgag
     2041 ctgctgacag aaaaagaggt tgctatgatt tttgtgaact ggaaggagct gattatgtgt
     2101 aatatcaaac tactaaaagc gctgagagtc cgcaagaaga tgtccgggga gaagatgcct
     2161 gtgaagatga ttggagacat cctgagcgca cagctgccgc acatgcagcc ctacatccgc
     2221 ttctgcagcc gccagctcaa cggggctgcc ctgatccagc agaagacgga cgaggcccca
     2281 gacttcaagg agttcgtcaa aagattggca atggatcctc ggtgtaaagg gatgccactc
     2341 tctagtttta tactgaagcc tatgcaacgg gtaacaagat acccactgat cattaaaaat
     2401 atcctggaaa acacccctga aaaccacccg gaccacagcc acttgaagca cgccctggag
     2461 aaggcggaag agctctgttc ccaggtgaac gaaggggtgc gggagaagga gaactctgac
     2521 cggctggagt ggatccaggc ccacgtgcag tgtgaaggcc tgtctgagca acttgtgttc
     2581 aattcagtga ccaattgctt ggggccgcgc aaatttctgc acagtgggaa gctctacaag
     2641 gccaagagca acaaggagct gtatggcttc cttttcaacg acttcctcct gctgactcag
     2701 atcacgaagc ctttggggtc ttctggcacc gacaaagtct tcagccccaa atcaaacctg
     2761 cagtataaaa tgtataaaac acctattttc ctaaatgagg ttctagtaaa attacccacc
     2821 gacccttctg gagacgagcc catcttccac atctcccaca ttgaccgcgt ctatactctc
     2881 cgagcagaaa gcataaatga aaggactgcc tgggtgcaga aaatcaaagc tgcttctgaa
     2941 ctctacatag agactgagaa aaagaagtaa gaattctgca gtcgacggta ccgcgggccc
     3001 gggatccacc ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     3061 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     3121 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3181 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3241 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     3301 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3361 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3421 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3481 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3541 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3601 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3661 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     3721 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3781 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3841 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3901 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3961 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4021 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4081 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4141 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4201 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     4261 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     4321 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     4381 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     4441 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     4501 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     4561 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     4621 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4681 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4741 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     4801 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4861 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4921 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4981 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     5041 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     5101 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     5161 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     5221 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     5281 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     5341 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     5401 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     5461 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     5521 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5581 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5641 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5701 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5761 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5821 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5881 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5941 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6001 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6061 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6121 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6181 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6241 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6301 gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
