LOCUS       sequence-346382-        6109 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 6109)
  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 6109)
  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-mCherry-DHPH-ITSN1
FEATURES             Location/Qualifiers
     source          1..6109
                     /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..1662
                     /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(1100..1118)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1368..1387
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2897..3018
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2934..2953)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2988..3007
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3025..3480)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3162..3183)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3374..3393
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3507..3611
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3613..3970
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3613..3804
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3636..3656)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3821..3956
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4005..4799
                     /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             4005..4799
                     /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(4059..4078)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4669..4688
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4987..5006)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5031..5078
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5407..5995
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5896..5915
                     /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 caccatggtg
      961 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     1021 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     1081 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     1141 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     1201 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     1261 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     1321 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     1381 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     1441 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     1501 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     1561 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     1621 gagggccgcc actccaccgg cggcatggac gagctgtaca agtccggact cagatctgat
     1681 atgttgaccc caactgaaag aaagcgacaa ggatacatcc acgagctcat tgtcaccgag
     1741 gagaactatg tgaatgacct gcagctggtc acagagattt ttcaaaaacc cctgatggag
     1801 tctgagctgc tgacagaaaa agaggttgct atgatttttg tgaactggaa ggagctgatt
     1861 atgtgtaata tcaaactact aaaagcgctg agagtccgca agaagatgtc cggggagaag
     1921 atgcctgtga agatgattgg agacatcctg agcgcacagc tgccgcacat gcagccctac
     1981 atccgcttct gcagccgcca gctcaacggg gctgccctga tccagcagaa gacggacgag
     2041 gccccagact tcaaggagtt cgtcaaaaga ttggcaatgg atcctcggtg taaagggatg
     2101 ccactctcta gttttatact gaagcctatg caacgggtaa caagataccc actgatcatt
     2161 aaaaatatcc tggaaaacac ccctgaaaac cacccggacc acagccactt gaagcacgcc
     2221 ctggagaagg cggaagagct ctgttcccag gtgaacgaag gggtgcggga gaaggagaac
     2281 tctgaccggc tggagtggat ccaggcccac gtgcagtgtg aaggcctgtc tgagcaactt
     2341 gtgttcaatt cagtgaccaa ttgcttgggg ccgcgcaaat ttctgcacag tgggaagctc
     2401 tacaaggcca agagcaacaa ggagctgtat ggcttccttt tcaacgactt cctcctgctg
     2461 actcagatca cgaagccttt ggggtcttct ggcaccgaca aagtcttcag ccccaaatca
     2521 aacctgcagt ataaaatgta taaaacacct attttcctaa atgaggttct agtaaaatta
     2581 cccaccgacc cttctggaga cgagcccatc ttccacatct cccacattga ccgcgtctat
     2641 actctccgag cagaaagcat aaatgaaagg actgcctggg tgcagaaaat caaagctgct
     2701 tctgaactct acatagagac tgagaaaaag aagtaagaat tctgcagtcg acggtaccgc
     2761 gggcccggga tccaccggat ctagataact gatcataatc agccatacca catttgtaga
     2821 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     2881 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2941 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3001 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     3061 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3121 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3181 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3241 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3301 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3361 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3421 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3481 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3541 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3601 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     3661 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3721 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3781 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3841 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3901 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3961 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4021 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4081 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4141 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     4201 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4261 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4321 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4381 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4441 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4501 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     4561 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4621 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4681 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4741 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4801 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4861 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4921 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     4981 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     5041 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     5101 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     5161 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     5221 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     5281 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5341 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5401 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5461 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5521 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5581 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5641 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5701 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5761 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5821 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5881 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5941 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6001 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6061 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
