LOCUS       sequence-346325-        5838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterodimerization with iLID, RhoA activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5838)
  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 5838)
  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-p63DH
FEATURES             Location/Qualifiers
     source          1..5838
                     /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"
     regulatory      1999..2008
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2626..2747
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2663..2682)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2717..2736
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2754..3209)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2891..2912)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3103..3122
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3236..3340
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3342..3699
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3342..3533
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3365..3385)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3550..3685
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3734..4528
                     /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             3734..4528
                     /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(3788..3807)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4398..4417
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4716..4735)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4760..4807
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5136..5724
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5625..5644
                     /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 tacaagtcca agaaggctct ggaaaggagt
     1921 atgtatgtcc tgagtgaact ggtagaaaca gagaaaatgt acgtggacga cttggggcag
     1981 attgtggagg gttatatggc caccatggct gctcaggggg tccccgagag tcttcgaggc
     2041 cgtgacagga ttgtgtttgg gaatatccag caaatctatg agtggcaccg agactatttc
     2101 ttgcaagagc tacaacggtg tctgaaagat cctgattggc tggctcagct attcatcaaa
     2161 cacgagcgcc ggctgcatat gtatgtggtg tactgtcaga ataagcccaa gtcagagcat
     2221 gtggtgtcag agtttgggga cagctacttt gaggagctcc ggcagcagct ggggcaccgc
     2281 ctgcagctga acgacctcct catcaaacct gtgcagcgga tcatgaaata ccagctgctg
     2341 ctcaaggatt ttctcaagta ttacaataga gctgggatgg atactgcaga cctagagcaa
     2401 gctgtggagg tcatgtgctt tgtgcccaag cgctgcaacg atatgatgac gctggggaga
     2461 ttgcggggat ttgagggcta aggtaccgcg ggcccgggat ccaccggatc tagataactg
     2521 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     2581 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     2641 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     2701 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt
     2761 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2821 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2881 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     2941 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3001 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3061 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3121 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3181 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     3241 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3301 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     3361 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3421 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3481 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3541 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3601 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     3661 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg
     3721 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3781 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3841 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3901 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3961 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4021 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4081 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4141 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4201 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4261 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4321 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4381 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4441 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4501 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4561 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4621 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4681 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg
     4741 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     4801 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     4861 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     4921 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt
     4981 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5041 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5101 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5161 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5221 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5281 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5341 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5401 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5461 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5521 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5581 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5641 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5701 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5761 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5821 cgtattaccg ccatgcat
//
