LOCUS       sequence-94069-e        6437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nuclear EGFP and Tomato in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6437)
  AUTHORS   Yoo AS, Staahl BT, Chen L, Crabtree GR
  TITLE     MicroRNA-mediated switching of chromatin-remodelling complexes in 
            neural development.
  JOURNAL   Nature. 2009 Jul 30;460(7255):642-6. doi: 10.1038/nature08139. Epub 
            2009 Jun 28.
  PUBMED    19561591
REFERENCE   2  (bases 1 to 6437)
  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: 
            "10.1038/nature08139"; journalName: "Nature"; date: "2009-07-30- 
            30"; volume: "460"; issue: "7255"; pages: "642-6"
COMMENT     Sequence Label: pNSEN-d2
FEATURES             Location/Qualifiers
     source          1..6437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        80..396
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      247..382
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      439..448
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             460..480
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             484..504
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             508..528
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(538..540,541..543,544..1254,1255..1260,1261..1383)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to residues 422-461 
                     of mouse ornithine decarboxylase, giving an in vivo 
                     half-life of ~2 hours"
                     /label=d2EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(583..604)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(844..863)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1191..1212
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1505..1586
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        1634..2013
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2014..2217
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2168..2188
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2262..2281
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2262..2280
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2309..2318
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2315..3016
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(2448..2466)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2716..2735
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2910..2933
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3026..3046
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3050..3070
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3074..3094
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3225..3346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3316..3335
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3353..3808)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3490..3511)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3702..3721
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3835..3939
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3941..4298
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3941..4132
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3964..3984)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4149..4284
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4333..5127
                     /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             4333..5127
                     /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(4387..4406)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4997..5016
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5315..5334)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5359..5406
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5735..6323
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6224..6243
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattac tagcgctacc ggactcagat ctgttaggcg ttttgcgctg cttcgcgaaa
       61 acgcgcgaga cgaaagattg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
      121 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
      181 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
      241 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
      301 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
      361 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tagcttgcat gcctgcaggt
      421 cggccgccac gaccggtcgc caccatggtg cacgtggatc caaaaaagaa gagaaaggta
      481 gatccaaaaa agaagagaaa ggtagatcca aaaaagaaga gaaaggtaca cgtgtccatg
      541 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      601 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      661 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      721 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      781 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      841 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      901 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      961 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1021 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1081 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1141 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1201 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagaagctt
     1261 agccatggct tcccgccgga ggtggaggag caggatgatg gcacgctgcc catgtcttgt
     1321 gcccaggaga gcgggatgga ccgtcaccct gcagcctgtg cttctgctag gatcaatgtg
     1381 taggcggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     1441 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     1501 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1561 cacaaataaa gcattttttt cactgcaatc tcgtgatacg cctatcgcga tgtacgggcc
     1621 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     1681 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     1741 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     1801 acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac
     1861 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     1921 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     1981 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     2041 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     2101 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     2161 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
     2221 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
     2281 gagacccaag ccccgggatc caccggtcgc caccatggtg agcaagggcg aggaggtcat
     2341 caaagagttc atgcgcttca aggtgcgcat ggagggctcc atgaacggcc acgagttcga
     2401 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     2461 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt tcatgtacgg
     2521 ctccaaggcg tacgtgaagc accccgccga catccccgat tacaagaagc tgtccttccc
     2581 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg tgaccgtgac
     2641 ccaggactcc tccctgcagg acggcacgct gatctacaag gtgaagatgc gcggcaccaa
     2701 cttccccccc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga
     2761 gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc tgaagctgaa
     2821 ggacggcggc cactacctgg tggagttcaa gaccatctac atggccaaga agcccgtgca
     2881 actgcccggc tactactacg tggacaccaa gctggacatc acctcccaca acgaggacta
     2941 caccatcgtg gaacagtacg agcgctccga gggccgccac cacctgttcc tgtacggcat
     3001 ggacgagctg tacaaggccg tggatccaaa aaagaagaga aaggtagatc caaaaaagaa
     3061 gagaaaggta gatccaaaaa agaagagaaa ggtacacggc taagcggccg cgactctaga
     3121 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     3181 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     3241 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3301 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta
     3361 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     3421 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     3481 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     3541 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     3601 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     3661 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     3721 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     3781 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3841 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3901 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     3961 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     4021 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     4081 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     4141 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4201 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     4261 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     4321 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4381 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4441 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4501 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4561 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4621 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4681 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4741 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4801 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4861 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4921 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4981 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5041 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     5101 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5161 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5221 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5281 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     5341 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     5401 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     5461 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     5521 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     5581 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5641 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5701 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5761 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5821 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5881 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5941 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6001 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6061 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6121 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6181 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6241 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6301 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6361 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6421 gtattaccgc catgcat
//
