LOCUS       sequence-325395-        5967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Nup54 without the FG-Nup domain, with mEGFP 
            (missing the first 5 amino acids) at amino acid position 494 with a 
            rigid alpha helix of 5 amino acids at the carboxyl end of mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5967)
  AUTHORS   Pulupa J, Prior H, Johnson DS, Simon SM
  TITLE     Conformation of the nuclear pore in living cells is modulated by 
            transport state.
  JOURNAL   Elife. 2020 Dec 21;9. pii: 60654. doi: 10.7554/eLife.60654.
  PUBMED    33346731
REFERENCE   2  (bases 1 to 5967)
  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: "Elife."; 
            date: "2020-12-21"; volume: "9. pii: 60654. doi"; pages: " 
            10.7554/eLife.60654"
COMMENT     Sequence Label: Nup54 no FG-mEGFP494(0)
FEATURES             Location/Qualifiers
     source          1..5967
                     /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"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     primer_bind     complement(1868..1889)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2129..2148)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2288..2539
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     2476..2497
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2755..2876
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2792..2811)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2846..2865
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2883..3338)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3020..3041)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3232..3251
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3365..3469
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3471..3828
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3471..3662
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3494..3514)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3679..3814
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3863..4657
                     /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             3863..4657
                     /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(3917..3936)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4527..4546
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4845..4864)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4889..4936
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5265..5853
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5754..5773
                     /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 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tcccgcgaca tggcgcagtc caaccaactc atcaacactg ccagcgctct
      721 ttctgctcca acgctattgg gggatgagag agatgccatc ttggcaaagt ggaaccagtt
      781 gcaggccttc tggggaacag gaaaaggata tttcaataat aacattcctc cagtggagtt
      841 tacacaggaa aacccctttt gccgatttaa ggctgtcggt tacagttgta tgcccaataa
      901 caaagatgaa gatggactag tggttctaat tttcaacaaa aaagaaacag atattagaag
      961 ccaacaacag cagttggtag aatcattgca taaagttctg ggaggaaacc agaccctcac
     1021 tgtcaatgtg gagggcatta agacactgcc cgacgatcag acagaagttg tcatttacat
     1081 tgttgagcgc tctccaaatg gcacctcgag aagagttcca gcgacaacgc tgtatgccca
     1141 ctttgagcaa gccaacatta aaacacagct tcagcagctt ggcgtgaccc tttctatgac
     1201 cagaacagag ctgtctcctg ctcagatcaa gcagctccta cagaaccctc ctgctggtgt
     1261 tgatcccatt atttgggaac aggccaaggt ggataacccg gattctgaaa aattaattcc
     1321 tgttccaatg gtgggtttca aagaattgct tcgaagactg aaggttcaag accagatgac
     1381 taagcagcac cagaccagat tagatatcat atctgaagat attagtgaac tgcaaaagaa
     1441 tcaaactaca actatggcca aaatagcaca atacaagagg aaactgatgg atctctcaca
     1501 cagaacctta caggtcctaa tcaagcagga aatccagagg aagagtggct atgccatcca
     1561 ggctgaggaa gaacagttgc gggttcagct agatacgatt caaggagagc tgaatgcacc
     1621 cactcagttc aaaggtcgac tgaatgaact gatgtcccag atcaggatgc agaatcattt
     1681 tggagctgtg aaatctgaag aaaagtatta catagatgca gacctattac gagagatcaa
     1741 gcagcatttg aaacagcaac aggaaggcct tagccacctg attagcatca taaaagacga
     1801 cctggaagat atcaagctgg tagaagctgc tgcaaaggag gagctgttca ccggggtggt
     1861 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1921 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1981 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     2041 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2101 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2161 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2221 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2281 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     2341 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2401 cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa
     2461 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2521 catggacgag ctgtacaagg agcatggact gaatgagacc atccacagca gaggcggggt
     2581 ctttagctga ccattgcgct gtccagaggt tcataaagct gtgtacaagt aaagcggccg
     2641 cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     2701 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     2761 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     2821 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     2881 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     2941 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3001 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3061 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3121 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3181 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3241 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3301 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     3361 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3421 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     3481 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3541 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3601 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3661 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3721 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3781 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     3841 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3901 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3961 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4021 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4081 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4141 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4201 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4261 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4321 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4381 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4441 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4501 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4561 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4621 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4681 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4741 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4801 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     4861 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     4921 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     4981 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     5041 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     5101 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5161 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5221 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5281 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5341 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5401 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5461 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5521 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5581 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5641 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5701 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5761 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5821 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5881 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5941 gtggataacc gtattaccgc catgcat
//
