LOCUS       sequence-354858-        4820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express SECFP with plasma membrane-targeting sequence in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4820)
  AUTHORS   Kashiwagi S, Fujioka Y, Satoh AO, Yoshida A, Fujioka M, Nepal P, 
            Tsuzuki A, Aoki O, Paudel S, Sasajima H, Ohba Y
  TITLE     Folding Latency of Fluorescent Proteins Affects the Mitochondrial 
            Localization of Fusion Proteins.
  JOURNAL   Cell Struct Funct. 2019 Dec 26;44(2):183-194. doi: 
            10.1247/csf.19028. Epub 2019 Nov 15.
  PUBMED    31735740
REFERENCE   2  (bases 1 to 4820)
  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: "10.1247/csf.19028";
            journalName: "Cell Struct Funct"; date: "2019-12-26- 26"; volume: 
            "44"; issue: "2"; pages: "183-194"
COMMENT     Sequence Label: pFX-SECFP-KRasCT
FEATURES             Location/Qualifiers
     source          1..4820
                     /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"
     CDS             join(630..632,633..635,636..1346)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(675..696)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(936..955)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1283..1304
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1608..1729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1645..1664)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1699..1718
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1736..2191)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1873..1894)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2085..2104
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2218..2322
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2324..2681
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2324..2515
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2347..2367)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2532..2667
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2716..3510
                     /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             2716..3510
                     /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(2770..2789)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3380..3399
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3698..3717)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3742..3789
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4118..4706
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4607..4626
                     /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 ccggttctag agccatggaa ttcggatcca tggtgagcaa gggcgaggag ctgttcaccg
      661 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacagg ttcagcgtgt
      721 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      781 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctgg ggcgtgcagt
      841 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
      901 aaggctacgt ccaggagcgt accatcttct tcaaggacga cggcaactac aagacccgcg
      961 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcggct
     1021 tcaaggagga cggcaacatc ctggggcaca agctagagta caactacatc agccacaacg
     1081 tctatatcac cgccgacaag cagaagaacg gcatcaaggc ccacttcaag atccgccaca
     1141 acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1201 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1261 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1321 ctctcggcat ggacgagctg tacaagggag gatctggagg aagatctgga ggatccggag
     1381 gactcgagag cggccgcaag atgagcaaag atggtaaaaa gaagaaaaag aagtcaaaga
     1441 caaagtgtgt aattatgtaa gggccgctga ctgactgaag atccaccgga tctagataac
     1501 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     1561 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     1621 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1681 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     1741 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     1801 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     1861 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     1921 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     1981 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2041 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     2101 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     2161 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     2221 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2281 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     2341 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2401 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2461 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2521 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2581 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     2641 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     2701 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     2761 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     2821 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     2881 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     2941 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3001 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3061 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3121 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3181 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3241 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3301 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3361 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3421 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3481 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3541 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3601 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3661 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     3721 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     3781 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     3841 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     3901 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     3961 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4021 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4081 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4141 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4201 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4261 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4321 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4381 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4441 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4501 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4561 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4621 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4681 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4741 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4801 accgtattac cgccatgcat
//
