LOCUS       sequence-224714-        7005 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  anchor iLID domain to plasma membrane with slower diffusion .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7005)
  AUTHORS   Van Geel O, Hartsuiker R, Gadella TWJ
  TITLE     Increasing spatial resolution of photoregulated GTPases through 
            immobilized peripheral membrane proteins.
  JOURNAL   Small GTPases. 2018 Sep 5:1-10. doi: 10.1080/21541248.2018.1507411.
  PUBMED    30182785
REFERENCE   2  (bases 1 to 7005)
  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: "Small 
            GTPases. 2018 Sep 5:1-10. doi: 10.1080/21541248.2018.1507411."
COMMENT     Sequence Label: EB3-eGFP-2xC2-iLID
FEATURES             Location/Qualifiers
     source          1..7005
                     /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"
     misc_feature    1440..1492
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1500..1509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1506..1508,1509..1511,1512..2222)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1506..1508,1509..1511,1512..2222)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1551..1572)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1812..1831)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2159..2180
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3222..3659
                     /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"
     polyA_signal    3793..3914
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3830..3849)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3884..3903
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3921..4376)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4058..4079)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4270..4289
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4403..4507
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4509..4866
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4509..4700
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4532..4552)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4717..4852
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4901..5695
                     /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             4901..5695
                     /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(4955..4974)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5565..5584
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5883..5902)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5927..5974
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6303..6891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6792..6811
                     /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 gctagcatgg
      601 ccgtcaatgt gtactccaca tctgtgacca gtgaaaatct gagtcgccat gatatgcttg
      661 catgggtcaa cgactccctg cacctcaact ataccaagat agaacagctt tgttcagggg
      721 cagcctactg ccagttcatg gacatgctct tccccggctg tgtgcacttg aggaaagtga
      781 agttccaggc caaactagag catgaataca tccacaactt caaggtgctg caagcagctt
      841 tcaagaagat gggtgttgac aaaatcattc ctgtagagaa attagtgaaa ggaaaattcc
      901 aagataattt tgagtttatt cagtggttta agaaattctt tgacgcaaac tatgatggaa
      961 aggattacaa ccctctgctg gcgcggcagg gccaggacgt agcgccacct cctaacccag
     1021 gtgatcagat cttcaacaaa tccaagaaac tcattggcac agcagttcca cagaggacgt
     1081 cccccacagg cccaaaaaac atgcagacct ctggccggct gagcaatgtg gcccccccct
     1141 gcattctccg gaagaatcct ccatcagccc gaaatggcgg ccatgagact gatgcccaaa
     1201 ttcttgaact caaccaacag ctggtggact tgaagctgac agtggatggg ctggagaagg
     1261 aacgtgactt ctacttcagc aaacttcgtg acatcgagct catctgccag gagcatgaaa
     1321 gtgaaaacag ccctgttatc tcaggcatca ttggcatcct ctatgccaca gaggaaggat
     1381 tcgcaccccc tgaggacgat gagattgaag agcatcaaca agaagaccag gacgagtacc
     1441 tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggtcg
     1501 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1561 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1621 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1681 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1741 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1801 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1861 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1921 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1981 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2041 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2101 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2161 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2221 agtccggaag tagttgcact gaacccctag gcctgaagga taataccatc cccaacaagc
     2281 agatcacagc ctccagctac tacaaaacct ggggcctgag tgcctttagc tggtttccct
     2341 actacgcacg actggataat cagggcaagt tcaacgcctg gaccgcccag accaacagtg
     2401 cctctgagtg gctgcagatt gacctgggct cccagaagcg agtcacgggc atcatcaccc
     2461 agggtgcccg agactttggc cacattcaat atgtggctgc ctacagggtg gcctatggtg
     2521 atgatggtgt gacctggact gagtacaagg acccgggggc ctcagaaagc aagattttcc
     2581 ctggtaacat ggacaataat tcccacaaga agaacatatt tgagacgccg ttccaggctc
     2641 gcttcgtgcg gatccagccc gtggcctggc acaaccgtat caccctgcga gtggagctgc
     2701 tgggctgtgg taagcttaga tcttgcactg aacccctagg cctgaaggat aataccatcc
     2761 ccaacaagca gatcacagcc tccagctact acaaaacctg gggcctgagt gcctttagct
     2821 ggtttcccta ctacgcacga ctggataatc agggcaagtt caacgcctgg accgcccaga
     2881 ccaacagtgc ctctgagtgg ctgcagattg acctgggctc ccagaagcga gtcacgggca
     2941 tcatcaccca gggtgcccga gactttggcc acattcaata tgtggctgcc tacagggtgg
     3001 cctatggtga tgatggtgtg acctggactg agtacaagga cccgggggcc tcagaaagca
     3061 agattttccc tggtaacatg gacaataatt cccacaagaa gaacatattt gagacgccgt
     3121 tccaggctcg cttcgtgcgg atccagcccg tggcctggca caaccgtatc accctgcgag
     3181 tggagctgct gggctgttat gaattcagca tgggggagtt tctggcaacc acactggaac
     3241 ggatcgagaa aaatttcgtg attactgatc cgagactgcc tgacaaccca atcatttttg
     3301 cgagcgattc cttcctgcag ctgacagaat attctcggga agagatcctg gggcgcaatt
     3361 gccgttttct gcagggaccc gagacagacc gtgccactgt tcggaaaatc agagatgcta
     3421 ttgacaacca gactgaagtg accgttcagc tgatcaatta taccaagagc ggcaagaagt
     3481 tctggaacgt gttccacctg cagccgatgc gcgattataa gggcgacgtc cagtacttca
     3541 ttggcgtgca gctggatggc accgaacgtc ttcatggcgc cgctgagcgt gaggcggtct
     3601 gcctgatcaa aaagacagcc tttcagattg ctgaggcagc gaacgacgaa aattactttg
     3661 gtacaagtaa agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     3721 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     3781 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3841 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3901 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     3961 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     4021 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     4081 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     4141 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     4201 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     4261 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     4321 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     4381 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4441 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4501 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     4561 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4621 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4681 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4741 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4801 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4861 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     4921 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     4981 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     5041 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     5101 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     5161 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     5221 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     5281 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     5341 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     5401 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     5461 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     5521 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     5581 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     5641 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     5701 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     5761 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5821 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     5881 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     5941 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     6001 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     6061 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     6121 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     6181 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6241 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6301 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6361 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6421 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6481 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6541 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6601 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6661 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6721 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6781 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6841 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6901 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6961 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
