LOCUS       sequence-224807-        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"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    245..301
                     /label=MCS
                     /note="multiple cloning site"
     CDS             377..706
                     /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             797..1126
                     /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      1139..1148
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1145..1840
                     /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(1278..1296)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1546..1565
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1913..2242
                     /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    3397..3518
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3434..3453)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3488..3507
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3525..3980)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3662..3683)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3874..3893
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4007..4111
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4113..4470
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4113..4304
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4136..4156)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4321..4456
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4505..5299
                     /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             4505..5299
                     /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(4559..4578)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5169..5188
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5487..5506)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5531..5578
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5907..6495
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6396..6415
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6670..6973
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 actcagatct cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc
      301 catgggactc agatctcgag ctcaagcttc gaacgaattc ggcattgatc tgagcggcct
      361 gaccctgcag gaattcagct ccccgaaacg ccctaagctg ctgcgtgaat attacgattg
      421 gctggttgat aacagcttta ccccatatct ggtggtggat gccacatacc tgggcgtgaa
      481 cgtgcccgtg gagtatgtga aagacggtca gatcgtgctg aatctgtctg caagtgcgac
      541 cggcaacctg caactgacaa atgattttat ccagttcaac gcccgcttta agggcgtgtc
      601 tcgtgaactg tatatcccga tgggtgccgc tctggccatt tacgctcgcg agaacggcga
      661 tggtgtgatg ttcgaaccag aagaaatcta tgacgagctg aatattggtc caccgggatc
      721 catgggactc agatctcgag ctcaagcttc gaacgaattc ggcattgatc tgagcggcct
      781 gaccctgcag gaattcagct ccccgaaacg ccctaagctg ctgcgtgaat attacgattg
      841 gctggttgat aacagcttta ccccatatct ggtggtggat gccacatacc tgggcgtgaa
      901 cgtgcccgtg gagtatgtga aagacggtca gatcgtgctg aatctgtctg caagtgcgac
      961 cggcaacctg caactgacaa atgattttat ccagttcaac gcccgcttta agggcgtgtc
     1021 tcgtgaactg tatatcccga tgggtgccgc tctggccatt tacgctcgcg agaacggcga
     1081 tggtgtgatg ttcgaaccag aagaaatcta tgacgagctg aatattggtc caccggtcgc
     1141 caccatggtg agcaagggcg aggcagtgat caaggagttc atgcggttca aggtgcacat
     1201 ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     1261 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttctcctg
     1321 ggacatcctg tcccctcagt tcatgtacgg ctccagggcc ttcaccaagc accccgccga
     1381 catccccgac tactataagc agtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     1441 cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc tccctggagg acggcaccct
     1501 gatctacaag gtgaagctcc gcggcaccaa cttccctcct gacggccccg taatgcagaa
     1561 gaagacaatg ggctgggaag cgtccaccga gcggttgtac cccgaggacg gcgtgctgaa
     1621 gggcgacatt aagatggccc tgcgcctgaa ggacggcggc cgctacctgg cggacttcaa
     1681 gaccacctac aaggccaaga agcccgtgca gatgcccggc gcctacaacg tcgaccgcaa
     1741 gttggacatc acctcccaca acgaggacta caccgtggtg gaacagtacg aacgctccga
     1801 gggccgccac tccaccggcg gcatggacga gctgtacaag ggactcagat ctcgagctca
     1861 agcttcgaac gaattcggca ttgatctgag cggcctgacc ctgcaggaat tcagctcccc
     1921 gaaacgccct aagctgctgc gtgaatatta cgattggctg gttgataaca gctttacccc
     1981 atatctggtg gtggatgcca catacctggg cgtgaacgtg cccgtggagt atgtgaaaga
     2041 cggtcagatc gtgctgaatc tgtctgcaag tgcgaccggc aacctgcaac tgacaaatga
     2101 ttttatccag ttcaacgccc gctttaaggg cgtgtctcgt gaactgtata tcccgatggg
     2161 tgccgctctg gccatttacg ctcgcgagaa cggcgatggt gtgatgttcg aaccagaaga
     2221 aatctatgac gagctgaata ttggtggatc catgggactc agatctcgag ctcaagcttc
     2281 gaacgaattc ggcattgatc tgagcggcct gaccctgcag gtccggaata tgttgacccc
     2341 aactgaaaga aagcgacaag gatacatcca cgagctcatt gtcaccgagg agaactatgt
     2401 gaatgacctg cagctggtca cagagatttt tcaaaaaccc ctgatggagt ctgagctgct
     2461 gacagaaaaa gaggttgcta tgatttttgt gaactggaag gagctgatta tgtgtaatat
     2521 caaactacta aaagcgctga gagtccgcaa gaagatgtcc ggggagaaga tgcctgtgaa
     2581 gatgattgga gacatcctga gcgcacagct gccgcacatg cagccctaca tccgcttctg
     2641 cagccgccag ctcaacgggg ctgccctgat ccagcagaag acggacgagg ccccagactt
     2701 caaggagttc gtcaaaagat tggcaatgga tcctcggtgt aaagggatgc cactctctag
     2761 ttttatactg aagcctatgc aacgggtaac aagataccca ctgatcatta aaaatatcct
     2821 ggaaaacacc cctgaaaacc acccggacca cagccacttg aagcacgccc tggagaaggc
     2881 ggaagagctc tgttcccagg tgaacgaagg ggtgcgggag aaggagaact ctgaccggct
     2941 ggagtggatc caggcccacg tgcagtgtga aggcctgtct gagcaacttg tgttcaattc
     3001 agtgaccaat tgcttggggc cgcgcaaatt tctgcacagt gggaagctct acaaggccaa
     3061 gagcaacaag gagctgtatg gcttcctttt caacgacttc ctcctgctga ctcagatcac
     3121 gaagcctttg gggtcttctg gcaccgacaa agtcttcagc cccaaatcaa acctgcagta
     3181 taaaatgtat aaaacaccta ttttcctaaa tgaggttcta gtaaaattac ccaccgaccc
     3241 ttctggagac gagcccatct tccacatctc ccacattgac cgcgtctata ctctccgagc
     3301 agaaagcata aatgaaagga ctgcctgggt gcagaaaatc aaagctgctt ctgaactcta
     3361 catagagact gagaaaaaga aggaattgtt gttgttaact tgtttattgc agcttataat
     3421 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3481 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
     3541 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     3601 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     3661 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     3721 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     3781 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     3841 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     3901 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     3961 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4021 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4081 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     4141 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4201 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4261 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4321 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4381 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4441 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     4501 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4561 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4621 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4681 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4741 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4801 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4861 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4921 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4981 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     5041 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     5101 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     5161 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     5221 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     5281 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     5341 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5401 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5461 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     5521 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     5581 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     5641 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     5701 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     5761 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5821 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5881 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5941 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6001 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6061 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6121 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6181 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6241 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6301 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6361 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6421 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6481 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6541 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6601 gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     6661 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     6721 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     6781 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     6841 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     6901 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     6961 cgctattacc atg
//
