LOCUS       sequence-122872-        4764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EBFP2 blue fluorescent protein with NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4764)
  AUTHORS   Ai HW, Shaner NC, Cheng Z, Tsien RY, Campbell RE
  TITLE     Exploration of New Chromophore Structures Leads to the 
            Identification of Improved Blue Fluorescent Proteins.
  JOURNAL   Biochemistry. 2007 May 22;46(20):5904-10. doi: 10.1021/bi700199g
  PUBMED    17444659
REFERENCE   2  (bases 1 to 4764)
  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"; journalName: 
            "Biochemistry"; date: "2007-05-22- 22"; volume: "46"; issue: "20"; 
            pages: "5904-10"
COMMENT     Sequence Label: pEBFP2-Nuc
FEATURES             Location/Qualifiers
     source          1..4764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        372..575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     526..546
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      614..623
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(620..622,623..625,626..1336)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(665..686)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             1361..1381
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1385..1405
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1409..1429
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1559..1680
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1596..1615)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1650..1669
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1687..2142)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1824..1845)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2036..2055
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2169..2273
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2275..2632
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2275..2466
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2298..2318)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2483..2618
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2667..3461
                     /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             2667..3461
                     /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(2721..2740)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3331..3350
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3649..3668)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3693..3740
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4069..4657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4558..4577
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
       61 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      121 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      181 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      241 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      301 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      361 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      421 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      481 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      541 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      601 agcgctaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
      661 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtga ggggcgaggg
      721 cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
      781 gcccgtgccc tggcccaccc tcgtgaccac cctgagccac ggcgtgcagt gcttcgcccg
      841 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
      901 ccaggagcgc accatcttct tcaaggacga cggcacctac aagacccgcg ccgaggtgaa
      961 gttcgagggc gacaccctag tgaaccgcat cgagctgaag ggcgtcgact tcaaggagga
     1021 cggcaacatc ctggggcaca agctggagta caacttcaac agccacaaca tctatatcat
     1081 ggccgtcaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acgtggagga
     1141 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1201 gctgctgccc gacagccact acctgagcac ccagtccgtg ctgagcaaag accccaacga
     1261 gaagcgcgat cacatggtcc tgctggagtt ccgcaccgcc gccgggatca ctctcggcat
     1321 ggacgagctg tacaagtccg gactcagatc tcgagctgat ccaaaaaaga agagaaaggt
     1381 agatccaaaa aagaagagaa aggtagatcc aaaaaagaag agaaaggtag gatccaccgg
     1441 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     1501 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     1561 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     1621 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     1681 aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     1741 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     1801 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     1861 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     1921 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     1981 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2041 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2101 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     2161 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2221 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     2281 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2341 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2401 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2461 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2521 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     2581 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     2641 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     2701 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     2761 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     2821 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     2881 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2941 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3001 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3061 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3121 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3181 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3241 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3301 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3361 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3421 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     3481 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     3541 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3601 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     3661 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     3721 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     3781 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     3841 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     3901 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3961 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4021 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4081 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4141 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4201 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4261 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4321 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4381 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4441 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4501 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4561 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4621 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4681 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4741 ttctgtggat aaccgtatta ccgc
//
