LOCUS       sequence-236268-        6157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 3xEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6157)
  AUTHORS   Bohm J, Thavaraja R, Giehler S, Nalaskowski MM
  TITLE     A set of enhanced green fluorescent protein concatemers for 
            quantitative determination of nuclear localization signal strength.
  JOURNAL   Anal Biochem. 2017 Sep 15;533:48-55. doi: 10.1016/j.ab.2017.06.015. 
            Epub 2017 Jun 30.
  PUBMED    28669708
REFERENCE   2  (bases 1 to 6157)
  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: "Anal 
            Biochem."; date: "2017-09-15"; volume: "533:48-55. doi"; pages: " 
            10.1016/j.ab.2017.06.015. Epub 2017 Jun 30"
COMMENT     Sequence Label: p3xEGFP-N1
FEATURES             Location/Qualifiers
     source          1..6157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      309..318
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(315..317,318..320,321..1031)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(315..317,318..320,321..1031)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    1858..1979
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1895..1914)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1949..1968
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1986..2441)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2123..2144)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2335..2354
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2468..2572
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2574..2931
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2574..2765
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2597..2617)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2782..2917
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2966..3760
                     /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
                     VLFVKTNLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3020..3039)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3630..3649
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3948..3967)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3992..4039
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4368..4956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4857..4876
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5131..5434
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5435..5638
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5589..5609
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      5734..5743
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5740..5742,5743..5745,5746..6157,1..299)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5740..5742,5743..5745,5746..6157,1..299)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
ORIGIN
        1 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
       61 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
      121 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
      181 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
      241 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtayaagg
      301 gtccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      361 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      421 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      481 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      541 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      601 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      661 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      721 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
      781 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
      841 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
      901 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
      961 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1021 agctgtacaa ggggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1081 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1141 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1201 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1261 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1321 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1381 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1441 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1501 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1561 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1621 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1681 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg
     1741 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     1801 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     1861 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     1921 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     1981 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2041 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2101 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2161 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     2221 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     2281 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     2341 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     2401 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     2461 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2521 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     2581 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2641 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2701 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2761 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     2821 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     2881 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     2941 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3001 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3061 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccaa
     3121 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     3181 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3241 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3301 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3361 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3421 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3481 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     3541 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3601 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3661 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     3721 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     3781 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     3841 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     3901 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     3961 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     4021 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     4081 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     4141 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     4201 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4261 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4321 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4381 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4441 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4501 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4561 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4621 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4681 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4741 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4801 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4861 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4921 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4981 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5041 tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc
     5101 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     5161 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     5221 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     5281 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     5341 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     5401 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     5461 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     5521 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     5581 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
     5641 tttagtgaac cgtcagatcc gctagcgcta ccggactcag atctcgagct caagcttgaa
     5701 atcgaattcc cgcgggcccg ggatccaccg gtcgccacca tggtgagcaa gggcgaggag
     5761 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     5821 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     5881 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     5941 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     6001 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     6061 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     6121 ggcatcgact tcaaggagga cggcaacatc ctggggc
//
