LOCUS       sequence-215999-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Translocating ITSN1 to iLID and activate Cdc42.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6973)
  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 6973)
  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: 2xsspB-mSc-sspB-ITSN1
FEATURES             Location/Qualifiers
     source          1..6973
                     /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    609..665
                     /label=MCS
                     /note="multiple cloning site"
     CDS             741..1070
                     /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"
     CDS             1161..1490
                     /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      1503..1512
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1509..2204
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1642..1660)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1910..1929
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2277..2606
                     /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"
     polyA_signal    3761..3882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3798..3817)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3852..3871
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3889..4344)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4026..4047)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4238..4257
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4371..4475
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4477..4834
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4477..4668
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4500..4520)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4685..4820
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4869..5663
                     /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             4869..5663
                     /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(4923..4942)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5533..5552
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5851..5870)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5895..5942
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6271..6859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6760..6779
                     /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 gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccatggg actcagatct cgagctcaag cttcgaacga attcggcatt gatctgagcg
      721 gcctgaccct gcaggaattc agctccccga aacgccctaa gctgctgcgt gaatattacg
      781 attggctggt tgataacagc tttaccccat atctggtggt ggatgccaca tacctgggcg
      841 tgaacgtgcc cgtggagtat gtgaaagacg gtcagatcgt gctgaatctg tctgcaagtg
      901 cgaccggcaa cctgcaactg acaaatgatt ttatccagtt caacgcccgc tttaagggcg
      961 tgtctcgtga actgtatatc ccgatgggtg ccgctctggc catttacgct cgcgagaacg
     1021 gcgatggtgt gatgttcgaa ccagaagaaa tctatgacga gctgaatatt ggtccaccgg
     1081 gatccatggg actcagatct cgagctcaag cttcgaacga attcggcatt gatctgagcg
     1141 gcctgaccct gcaggaattc agctccccga aacgccctaa gctgctgcgt gaatattacg
     1201 attggctggt tgataacagc tttaccccat atctggtggt ggatgccaca tacctgggcg
     1261 tgaacgtgcc cgtggagtat gtgaaagacg gtcagatcgt gctgaatctg tctgcaagtg
     1321 cgaccggcaa cctgcaactg acaaatgatt ttatccagtt caacgcccgc tttaagggcg
     1381 tgtctcgtga actgtatatc ccgatgggtg ccgctctggc catttacgct cgcgagaacg
     1441 gcgatggtgt gatgttcgaa ccagaagaaa tctatgacga gctgaatatt ggtccaccgg
     1501 tcgccaccat ggtgagcaag ggcgaggcag tgatcaagga gttcatgcgg ttcaaggtgc
     1561 acatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1621 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttct
     1681 cctgggacat cctgtcccct cagttcatgt acggctccag ggccttcacc aagcaccccg
     1741 ccgacatccc cgactactat aagcagtcct tccccgaggg cttcaagtgg gagcgcgtga
     1801 tgaacttcga ggacggcggc gccgtgaccg tgacccagga cacctccctg gaggacggca
     1861 ccctgatcta caaggtgaag ctccgcggca ccaacttccc tcctgacggc cccgtaatgc
     1921 agaagaagac aatgggctgg gaagcgtcca ccgagcggtt gtaccccgag gacggcgtgc
     1981 tgaagggcga cattaagatg gccctgcgcc tgaaggacgg cggccgctac ctggcggact
     2041 tcaagaccac ctacaaggcc aagaagcccg tgcagatgcc cggcgcctac aacgtcgacc
     2101 gcaagttgga catcacctcc cacaacgagg actacaccgt ggtggaacag tacgaacgct
     2161 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagggactc agatctcgag
     2221 ctcaagcttc gaacgaattc ggcattgatc tgagcggcct gaccctgcag gaattcagct
     2281 ccccgaaacg ccctaagctg ctgcgtgaat attacgattg gctggttgat aacagcttta
     2341 ccccatatct ggtggtggat gccacatacc tgggcgtgaa cgtgcccgtg gagtatgtga
     2401 aagacggtca gatcgtgctg aatctgtctg caagtgcgac cggcaacctg caactgacaa
     2461 atgattttat ccagttcaac gcccgcttta agggcgtgtc tcgtgaactg tatatcccga
     2521 tgggtgccgc tctggccatt tacgctcgcg agaacggcga tggtgtgatg ttcgaaccag
     2581 aagaaatcta tgacgagctg aatattggtg gtaccatggg actcagatct cgagctcaag
     2641 cttcgaacga attcggcatt gatctgagcg gcctgaccct gcaggtccgg aatatgttga
     2701 ccccaactga aagaaagcga caaggataca tccacgagct cattgtcacc gaggagaact
     2761 atgtgaatga cctgcagctg gtcacagaga tttttcaaaa acccctgatg gagtctgagc
     2821 tgctgacaga aaaagaggtt gctatgattt ttgtgaactg gaaggagctg attatgtgta
     2881 atatcaaact actaaaagcg ctgagagtcc gcaagaagat gtccggggag aagatgcctg
     2941 tgaagatgat tggagacatc ctgagcgcac agctgccgca catgcagccc tacatccgct
     3001 tctgcagccg ccagctcaac ggggctgccc tgatccagca gaagacggac gaggccccag
     3061 acttcaagga gttcgtcaaa agattggcaa tggatcctcg gtgtaaaggg atgccactct
     3121 ctagttttat actgaagcct atgcaacggg taacaagata cccactgatc attaaaaata
     3181 tcctggaaaa cacccctgaa aaccacccgg accacagcca cttgaagcac gccctggaga
     3241 aggcggaaga gctctgttcc caggtgaacg aaggggtgcg ggagaaggag aactctgacc
     3301 ggctggagtg gatccaggcc cacgtgcagt gtgaaggcct gtctgagcaa cttgtgttca
     3361 attcagtgac caattgcttg gggccgcgca aatttctgca cagtgggaag ctctacaagg
     3421 ccaagagcaa caaggagctg tatggcttcc ttttcaacga cttcctcctg ctgactcaga
     3481 tcacgaagcc tttggggtct tctggcaccg acaaagtctt cagccccaaa tcaaacctgc
     3541 agtataaaat gtataaaaca cctattttcc taaatgaggt tctagtaaaa ttacccaccg
     3601 acccttctgg agacgagccc atcttccaca tctcccacat tgaccgcgtc tatactctcc
     3661 gagcagaaag cataaatgaa aggactgcct gggtgcagaa aatcaaagct gcttctgaac
     3721 tctacataga gactgagaaa aagaaggaat tgttgttgtt aacttgttta ttgcagctta
     3781 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3841 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg
     3901 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3961 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     4021 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     4081 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     4141 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     4201 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     4261 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     4321 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     4381 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     4441 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     4501 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4561 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     4621 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     4681 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4741 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4801 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     4861 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4921 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4981 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     5041 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     5101 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     5161 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     5221 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     5281 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     5341 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     5401 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     5461 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     5521 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     5581 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     5641 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     5701 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5761 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5821 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     5881 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     5941 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     6001 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     6061 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     6121 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6181 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6241 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6301 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6361 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     6421 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6481 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6541 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6601 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6661 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6721 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6781 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6841 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6901 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6961 taccgccatg cat
//
