LOCUS       sequence-236201-        5456 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 2xEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5456)
  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 5456)
  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: p2xEGFP-N1
FEATURES             Location/Qualifiers
     source          1..5456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      383..392
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(389..391,392..394,395..1108)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(389..391,392..394,395..1105)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    1231..1352
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1268..1287)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1322..1341
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1359..1814)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1496..1517)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1708..1727
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1841..1945
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1947..2304
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1947..2138
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1970..1990)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2155..2290
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2339..3133
                     /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             2339..3133
                     /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(2393..2412)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3003..3022
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3321..3340)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3365..3412
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3741..4329
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4230..4249
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4504..4807
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4808..5011
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4962..4982
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      5107..5116
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5113..5115,5116..5118,5119..5456,1..373)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5113..5115,5116..5118,5119..5456,1..373)
                     /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 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
       61 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
      121 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
      181 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
      241 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
      301 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
      361 cgagctgtat aagggtccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
      421 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      481 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      541 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
      601 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      661 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
      721 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
      781 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
      841 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
      901 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
      961 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     1021 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1081 tctcggcatg gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat
     1141 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     1201 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     1261 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     1321 tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt
     1381 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     1441 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     1501 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     1561 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     1621 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     1681 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     1741 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     1801 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1861 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1921 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt
     1981 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2041 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2101 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2161 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2221 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     2281 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat
     2341 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     2401 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     2461 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     2521 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     2581 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     2641 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     2701 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     2761 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     2821 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     2881 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     2941 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3001 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3061 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3121 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     3181 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3241 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3301 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     3361 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc
     3421 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa
     3481 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg
     3541 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt
     3601 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3661 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3721 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3781 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3841 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     3901 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3961 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4021 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4081 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4141 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4201 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4261 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4321 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4381 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
     4441 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     4501 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     4561 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     4621 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     4681 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     4741 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     4801 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     4861 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     4921 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     4981 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg
     5041 ctaccggact cagatctcga gctcaagctt gaaatcgaat tcccgcgggc ccgggatcca
     5101 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     5161 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     5221 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     5281 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     5341 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     5401 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagtt
//
