LOCUS       sequence-377467-        5287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Visualizing endogenous.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5287)
  AUTHORS   Mahlandt EK, Kreider-Letterman G, Chertkova AO, Garcia-Mata R, 
            Goedhart J
  TITLE     Cell-based optimization and characterization of genetically encoded 
            location-based biosensors for Cdc42 or Rac activity.
  JOURNAL   J Cell Sci. 2023 May 15;136(10):jcs260802. doi: 10.1242/jcs.260802. 
            Epub 2023 May 25.
  PUBMED    37226883
REFERENCE   2  (bases 1 to 5287)
  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: "J Cell 
            Sci."; date: "2023-05-15"; volume: "136(10):jcs260802. doi"; pages: 
            " 10.1242/jcs.260802. Epub 2023 May 25"
COMMENT     Sequence Label: N-WASP(CRIB)-mScarlet-I-N-WASP(CRIB)
FEATURES             Location/Qualifiers
     source          1..5287
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        81..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..588
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     539..559
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      920..929
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             926..1621
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1059..1077)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1327..1346
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    1919..1971
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2095..2216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2132..2151)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2186..2205
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2223..2678)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2360..2381)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2572..2591
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2705..2809
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2811..3168
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2811..3002
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2834..2854)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3019..3154
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3203..3997
                     /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             3203..3997
                     /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(3257..3276)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3867..3886
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4185..4204)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4229..4276
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4605..5193
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5094..5113
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      421 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      481 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      541 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
      601 cgtcagatcc gctagcatga tctcccacac caaagaaaag aagaaaggaa aagctaaaaa
      661 gaagagatta accaaggcag atattggaac accaagtaat ttccagcaca ttgggcatgt
      721 tggatgggat ccaaatacag gttttgatct aaataatttg gatccagaat taaagaatct
      781 ttttgatatg tgtgggatct ctgaggccca gcttaaagac agagaaacat caaaagttat
      841 ttatgacttt attgaaaaaa caggaggtgt agaagctgtt aaaaacgaac tccgaagaca
      901 agcaccacca gcaccggtcg ccaccatggt gagcaagggc gaggcagtga tcaaggagtt
      961 catgcggttc aaggtgcaca tggagggctc catgaacggc cacgagttcg agatcgaggg
     1021 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     1081 tggccccctg cccttctcct gggacatcct gtcccctcag ttcatgtacg gctccagggc
     1141 cttcatcaag caccccgccg acatccccga ctactataag cagtccttcc ccgagggctt
     1201 caagtgggag cgcgtgatga acttcgagga cggcggcgcc gtgaccgtga cccaggacac
     1261 ctccctggag gacggcaccc tgatctacaa ggtgaagctc cgcggcacca acttccctcc
     1321 tgacggcccc gtaatgcaga agaagacaat gggctgggaa gcgtccaccg agcggttgta
     1381 ccccgaggac ggcgtgctga agggcgacat taagatggcc ctgcgcctga aggacggcgg
     1441 ccgctacctg gcggacttca agaccaccta caaggccaag aagcccgtgc agatgcccgg
     1501 cgcctacaac gtcgaccgca agttggacat cacctcccac aacgaggact acaccgtggt
     1561 ggaacagtac gaacgctccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     1621 gatgatctcc cacaccaaag aaaagaagaa aggaaaagct aaaaagaaga gattaaccaa
     1681 ggcagatatt ggaacaccaa gtaatttcca gcacattggg catgttggat gggatccaaa
     1741 tacaggtttt gatctaaata atttggatcc agaattaaag aatctttttg atatgtgtgg
     1801 gatctctgag gcccagctta aagacagaga aacatcaaaa gttatttatg actttattga
     1861 aaaaacagga ggtgtagaag ctgttaaaaa cgaactccga agacaagcac caccataact
     1921 cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc caccggatct
     1981 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     2041 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     2101 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     2161 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     2221 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     2281 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2341 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2401 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2461 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2521 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2581 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     2641 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     2701 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2761 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     2821 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2881 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2941 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3001 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3061 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3121 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     3181 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3241 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3301 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3361 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     3421 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3481 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3541 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3601 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3661 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3721 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3781 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3841 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3901 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     3961 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4021 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4081 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4141 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     4201 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     4261 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     4321 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     4381 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     4441 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4501 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4561 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4621 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4681 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4741 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4801 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4861 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4921 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4981 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5041 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5101 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5161 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5221 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5281 gtggata
//
