LOCUS       sequence-346326-        5261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasma membrane localized iLID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5261)
  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 5261)
  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: Lck-mTurquoise2-iLID
FEATURES             Location/Qualifiers
     source          1..5261
                     /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"
     regulatory      595..604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      640..649
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(646..648,649..651,652..1362)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(691..712)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(952..971)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1299..1320
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1384..1821
                     /codon_start=1
                     /product="improved light-inducible dimer when used with 
                     SspB nano or SspB micro (Guntas et al., 2015)"
                     /label=iLID
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNVFHLQPMRDYKGDVQ
                     YFIGVQLDGTERLHGAAEREAVCLIKKTAFQIAEAANDENYF"
     misc_feature    1873..1925
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2049..2170
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2086..2105)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2140..2159
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2177..2632)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2314..2335)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2526..2545
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2659..2763
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2765..3122
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2765..2956
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2788..2808)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2973..3108
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3157..3951
                     /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             3157..3951
                     /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(3211..3230)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3821..3840
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4139..4158)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4183..4230
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4559..5147
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5048..5067
                     /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 atgggctgcg tgtgcagcag caaccccgag ctaccggtcg ccaccatggt gagcaagggc
      661 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
      721 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
      781 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
      841 tcctggggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
      901 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
      961 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1021 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1081 tactttagcg acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac
     1141 ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgccgacca ctaccagcag
     1201 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1261 tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1321 accgccgccg ggatcactct cggcatggac gagctgtaca agggtagtgg tagtggggag
     1381 tttctggcaa ccacactgga acggatcgag aaaaatttcg tgattactga tccgagactg
     1441 cctgacaacc caatcatttt tgcgagcgat tccttcctgc agctgacaga atattctcgg
     1501 gaagagatcc tggggcgcaa ttgccgtttt ctgcagggac ccgagacaga ccgtgccact
     1561 gttcggaaaa tcagagatgc tattgacaac cagactgaag tgaccgttca gctgatcaat
     1621 tataccaaga gcggcaagaa gttctggaac gtgttccacc tgcagccgat gcgcgattat
     1681 aagggcgacg tccagtactt cattggcgtg cagctggatg gcaccgaacg tcttcatggc
     1741 gccgctgagc gtgaggcggt ctgcctgatc aaaaagacag cctttcagat tgctgaggca
     1801 gcgaacgacg aaaattactt tggaagcggg agtgggagca aaaagaagaa aaagaagtca
     1861 aagacaaagt aactcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
     1921 gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac
     1981 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     2041 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     2101 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     2161 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     2221 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2281 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2341 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2401 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2461 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2521 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2581 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     2641 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2701 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2761 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2821 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2881 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2941 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3001 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3061 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3121 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3181 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3241 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3301 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     3361 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3421 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3481 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3541 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3601 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3661 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     3721 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3781 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3841 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3901 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     3961 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4021 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4081 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     4141 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     4201 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     4261 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     4321 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     4381 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     4441 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4501 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4561 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4621 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4681 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4741 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4801 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4861 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4921 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4981 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5041 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5101 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5161 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5221 tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
