LOCUS       sequence-224586-        7054 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Translocating Tiam1 to iLID and activate Rac1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7054)
  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 7054)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Tiam1
FEATURES             Location/Qualifiers
     source          1..7054
                     /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    3478..3599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3515..3534)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3569..3588
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3606..4061)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3743..3764)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3955..3974
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4088..4192
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4194..4551
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4194..4385
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4217..4237)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4402..4537
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4586..5380
                     /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             4586..5380
                     /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(4640..4659)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5250..5269
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5568..5587)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5612..5659
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5988..6576
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6477..6496
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6751..7054
                     /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 ttggtggtac catgggactc agatctcgag ctcaagcttc
     2281 gaacgaattc ggcattgatc tgagcggcct gaccctgcag gtccggaata tgcgacagct
     2341 gtcggatgcg gataagctgc gcaaggtgat ctgtgaactg ctggagactg aacgcaccta
     2401 tgtgaaagac ttaaactgcc tcatggagag atacctgaag ccccttcaga aggagacctt
     2461 tctcacccag gatgagcttg atgtactttt tggaaattta accgaaatgg tggagtttca
     2521 agtcgagttc cttaagactc tagaagatgg agtaagactg gtccctgact tggaaaagct
     2581 ggagaaggtt gaccagttca agaaagtgct cttctctctg gggggatcct tcctgtacta
     2641 cgcggaccgc ttcaagctct acagtgcctt ctgtgcgagc cacacaaaag tccccaaggt
     2701 cctggtgaaa gccaagacgg acacagcctt caaggcgttc ctcgatgccc agaacccgag
     2761 gcagcagcac tcatccacgc tggagtctta cctcatcaag cccatccaga gggtcctcaa
     2821 gtacccgctt ctgctcaggg agctgtttgc gctgaccgat gcggagagcg aggaacacta
     2881 ccacctggac gtggccatca agactatgaa caaggtcgcc agtcacatca atgagatgca
     2941 gaagatccac gaagagtttg gtgccgtgtt tgaccagctg attgctgagc agacgggaga
     3001 gaagaaagag gttgcagatc tgagcatggg tgacctgctc ttgcacacca gcgtcatctg
     3061 gctgaaccca cccgcctcac tgggaaagtg gaaaaaggag ccagaattag cagccttcgt
     3121 cttcaaaacg gccgtggtcc ttgtatataa agacggttcc aagcagaaga agaaacttgt
     3181 tggctctcac aggctgtcaa tctacgagga gtgggacccc ttccggtttc gccacatgat
     3241 ccctaccgaa gctttgcagg tccgagctct gcccagtgca gatgcagagg caaatgcggt
     3301 atgcgaaatt gtccacgtga aatcagagtc agaagggagg cccgagcggg ttttccacct
     3361 ctgctgcagt tccccagaga gcagaaagga ctttctgaag tctgtgcatt cgatcctgcg
     3421 agataaacac agaagacagc tcctcaaaac ggaagaattc tgacaattgt tgttgttaac
     3481 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3541 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     3601 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     3661 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     3721 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     3781 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     3841 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     3901 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3961 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     4021 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     4081 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4141 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     4201 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4261 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     4321 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     4381 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4441 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     4501 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     4561 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4621 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4681 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4741 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     4801 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4861 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4921 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4981 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     5041 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     5101 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     5161 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     5221 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5281 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5341 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     5401 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     5461 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5521 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     5581 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     5641 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     5701 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     5761 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     5821 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5881 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5941 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6001 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6061 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6121 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6181 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6241 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6301 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6361 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6421 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6481 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6541 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6601 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6661 tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc
     6721 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     6781 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     6841 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     6901 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     6961 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     7021 gtacatctac gtattagtca tcgctattac catg
//
