LOCUS       sequence-329451-        5304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescently tagged GTPase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5304)
  AUTHORS   O'Shaughnessy EC, Stone OJ, LaFosse PK, Azoitei ML, Tsygankov D, 
            Heddleston JM, Legant WR, Wittchen ES, Burridge K, Elston TC, Betzig
            E, Chew TL, Adalsteinsson D, Hahn KM
  TITLE     Software for lattice light-sheet imaging of FRET biosensors, 
            illustrated with a new Rap1 biosensor.
  JOURNAL   J Cell Biol. 2019 Sep 2;218(9):3153-3160. doi: 
            10.1083/jcb.201903019. Epub 2019 Aug 23.
  PUBMED    31444239
REFERENCE   2  (bases 1 to 5304)
  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"; doi: 
            "10.1083/jcb.201903019"; journalName: "J Cell Biol"; date: 
            "2019-09-2- 2"; volume: "218"; issue: "9"; pages: "3153-3160"
COMMENT     Sequence Label: mCerulean3-Rap1
FEATURES             Location/Qualifiers
     source          1..5304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        175..478
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        479..682
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     633..653
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             714..737
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(747..749,750..752,753..1463)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(792..813)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1053..1072)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1400..1421
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2206..2327
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2243..2262)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2297..2316
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2334..2789)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2471..2492)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2683..2702
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2816..2920
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2922..3279
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2922..3113
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2945..2965)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3130..3265
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3314..4108
                     /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             3314..4108
                     /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(3368..3387)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3978..3997
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4296..4315)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4340..4387
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4716..5304
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5205..5224
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
      121 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      181 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      241 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      301 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      361 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      421 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      481 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      541 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      601 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      661 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc atggactata
      721 aggacgatga tgacaaaggt accggtatgg tgagcaaggg cgaggagctg ttcaccgggg
      781 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
      841 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
      901 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gtcctggggc gtgcagtgct
      961 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1021 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1081 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1141 aggaggacgg caacatcctg gggcacaagc tggagtacaa cgccatccac ggcaacgtct
     1201 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcggcctc aactgcaaca
     1261 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1321 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     1381 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1441 tcggcatgga cgagctgtac aagtccggac tcagatctcg agctaagctt atgcgtgagt
     1501 ataagctagt cgttcttggc tcaggaggcg ttggaaagtc tgctttgact gtacaatttg
     1561 ttcaaggaat ttttgtagaa aaatacgatc ctacgataga agattcttat agaaagcaag
     1621 ttgaagtaga tgcacaacag tgtatgcttg aaatcttgga tactgcagga acggagcaat
     1681 ttacagcaat gagggattta tacatgaaaa atggacaagg atttgcatta gtttattcca
     1741 tcacagcaca gtccacattt aacgatttac aagacctgag agaacagatt cttcgagtta
     1801 aagacactga tgatgttcca atgattcttg ttggtaataa gtgtgacttg gaagatgaaa
     1861 gagttgtagg gaaggaacaa ggtcaaaatc tagcaagaca atggaacaac tgtgcattct
     1921 tagaatcttc tgcaaaatca aaaataaatg ttaatgagat cttttatgac ctagtgcggc
     1981 aaattaacag aaaaactcca gtgcctggga aggctcgcaa aaagtcatca tgtcagctgc
     2041 tttaagaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggatc tagataactg
     2101 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     2161 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     2221 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     2281 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt
     2341 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2401 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2461 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     2521 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     2581 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     2641 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     2701 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     2761 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     2821 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2881 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     2941 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3001 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3061 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3121 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3181 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     3241 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg
     3301 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3361 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3421 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3481 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3541 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     3601 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     3661 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     3721 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     3781 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     3841 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     3901 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     3961 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4021 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4081 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4141 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4201 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4261 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg
     4321 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     4381 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     4441 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     4501 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt
     4561 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4621 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4681 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4741 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4801 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4861 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4921 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4981 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5041 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5101 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5161 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5221 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5281 tcaggggggc ggagcctatg gaaa
//
