LOCUS       sequence-346366-        6301 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 6301)
  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 6301)
  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-HaloTag-DHPH-ITSN1
FEATURES             Location/Qualifiers
     source          1..6301
                     /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"
     polyA_signal    3089..3210
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3126..3145)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3180..3199
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3217..3672)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3354..3375)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3566..3585
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3699..3803
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3805..4162
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3805..3996
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3828..3848)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4013..4148
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4197..4991
                     /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             4197..4991
                     /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(4251..4270)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4861..4880
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5179..5198)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5223..5270
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5599..6187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6088..6107
                     /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 caccatggcc
      961 gagatcggca ccggcttccc cttcgacccc cactacgtgg aggtgctggg cgagcgcatg
     1021 cactacgttg acgtgggccc ccgcgacggc acccccgtgc tgttcctgca cggcaacccc
     1081 accagcagct acgtgtggcg caacatcatc ccccacgtgg cccccaccca ccgcactatc
     1141 gcccccgacc tgatcggcat gggcaagagc gacaagcccg acctgggcta cttcttcgac
     1201 gaccacgtgc gcttcatgga cgccttcatc gaggccctgg gcctggagga ggtggtgctg
     1261 gtgatccacg actggggcag cgccctgggc ttccactggg ccaagcgcaa ccccgagcgc
     1321 gtgaagggca tcgccttcat ggagttcatc cgccccatcc ccacctggga cgagtggccc
     1381 gagttcgccc gcgagacctt ccaggccttc cgcaccaccg acgtgggccg caagctgatc
     1441 atcgaccaga acgtgttcat cgagggcacc ctgcccatgg gcgtggtgcg ccccctgacc
     1501 gaggtggaga tggaccacta ccgcgagccc ttcctgaacc ccgtggaccg cgagcccctg
     1561 tggcgcttcc ccaacgagct gcccatcgcc ggcgagcccg ccaacatcgt ggccctggtg
     1621 gaggagtaca tggactggct gcaccagagc cccgtgccca agctgctgtt ctggggcacc
     1681 cccggcgtgc tgatcccccc cgccgaggcc gcccgcctgg ccaagagcct gcccaacgtt
     1741 aaggccgtgg acatcggccc cggcctgaac ctgctccagg aggacaaccc cgacctgatc
     1801 ggcagcgaga tcgcccgctg gctgagcacc ctggagatca gcggcgtgta caagtccgga
     1861 ctcagatctg atatgttgac cccaactgaa agaaagcgac aaggatacat ccacgagctc
     1921 attgtcaccg aggagaacta tgtgaatgac ctgcagctgg tcacagagat ttttcaaaaa
     1981 cccctgatgg agtctgagct gctgacagaa aaagaggttg ctatgatttt tgtgaactgg
     2041 aaggagctga ttatgtgtaa tatcaaacta ctaaaagcgc tgagagtccg caagaagatg
     2101 tccggggaga agatgcctgt gaagatgatt ggagacatcc tgagcgcaca gctgccgcac
     2161 atgcagccct acatccgctt ctgcagccgc cagctcaacg gggctgccct gatccagcag
     2221 aagacggacg aggccccaga cttcaaggag ttcgtcaaaa gattggcaat ggatcctcgg
     2281 tgtaaaggga tgccactctc tagttttata ctgaagccta tgcaacgggt aacaagatac
     2341 ccactgatca ttaaaaatat cctggaaaac acccctgaaa accacccgga ccacagccac
     2401 ttgaagcacg ccctggagaa ggcggaagag ctctgttccc aggtgaacga aggggtgcgg
     2461 gagaaggaga actctgaccg gctggagtgg atccaggccc acgtgcagtg tgaaggcctg
     2521 tctgagcaac ttgtgttcaa ttcagtgacc aattgcttgg ggccgcgcaa atttctgcac
     2581 agtgggaagc tctacaaggc caagagcaac aaggagctgt atggcttcct tttcaacgac
     2641 ttcctcctgc tgactcagat cacgaagcct ttggggtctt ctggcaccga caaagtcttc
     2701 agccccaaat caaacctgca gtataaaatg tataaaacac ctattttcct aaatgaggtt
     2761 ctagtaaaat tacccaccga cccttctgga gacgagccca tcttccacat ctcccacatt
     2821 gaccgcgtct atactctccg agcagaaagc ataaatgaaa ggactgcctg ggtgcagaaa
     2881 atcaaagctg cttctgaact ctacatagag actgagaaaa agaagtaaga attctgcagt
     2941 cgacggtacc gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac
     3001 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     3061 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     3121 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3181 tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt
     3241 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     3301 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     3361 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     3421 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     3481 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     3541 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     3601 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     3661 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3721 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3781 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     3841 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3901 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3961 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4021 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4081 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4141 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     4201 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     4261 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4321 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     4381 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4441 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4501 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4561 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4621 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4681 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     4741 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4801 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4861 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4921 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4981 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     5041 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     5101 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5161 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     5221 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     5281 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     5341 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     5401 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     5461 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5521 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5581 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5641 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5701 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5761 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5821 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5881 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5941 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6001 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6061 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6121 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6181 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6241 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
     6301 t
//
