LOCUS       sequence-399866-        5958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nucleocytoplasmic transport probe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5958)
  TITLE     Roca-Cusachs Lab Plasmids 2023
REFERENCE   2  (bases 1 to 5958)
  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"
COMMENT     Sequence Label: H_NLS 80kDa
FEATURES             Location/Qualifiers
     source          1..5958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        51..354
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        355..558
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     509..529
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             609..629
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(645..647,648..1358)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(645..647,648..1358)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(687..708)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(948..967)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1295..1316
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2742..2759
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2812..2830)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2826..2873
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2899..2918
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2936..3391)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3073..3094)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3285..3304
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3418..3522
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3547..3567)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3591..3801
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3613..3809
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3660..3795
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3844..4638
                     /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             3844..4638
                     /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(3898..3917)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4508..4527
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4826..4845)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4870..4917
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5246..5834
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5735..5754
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
       61 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      121 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      181 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      241 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      301 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      361 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      421 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      481 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      541 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggtcgcca
      601 ccatgggccc aaaaaagaaa agaaaagttg gaggcggggg cagcgtgagc aagggcgagg
      661 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
      721 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
      781 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
      841 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
      901 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
      961 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1021 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1081 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1141 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1201 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     1261 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1321 ccgccgggat cactctcggc atggacgagc tgtacaagaa gcttgctcaa caaaatgctt
     1381 tttatcaagt cttaaatatg cctaacttaa atgctgatca acgcaatggt tttatccaaa
     1441 gccttaaaga tgatccaagc caaagtgcta acgttttagg tgaagctcaa aaacttaatg
     1501 actctcaagc tccaaaagct gatgcgcaac aaaataactt caacaaagat caacaaagcg
     1561 ccttctatga aatcttgaac atgcctaact taaacgaagc gcaacgtaac ggcttcattc
     1621 aaagtcttaa agacgaccca agccaaagca ctaacgtttt aggtgaagct aaaaaattaa
     1681 acgaatctca agcaccgaaa gctgataaca atttcaacaa agaacaacaa aatgctttct
     1741 atgaaatctt gaatatgcct aacttaaacg aagaacaacg caatggtttc atccaaagct
     1801 taaaagatga cccaagccaa agtgctaacc tattgtcaga agctaaaaag ttaaatgaat
     1861 ctcaagcacc gaaagcggat aacaaattca acaaagaaca acaaaatgct ttctatgaaa
     1921 tcttacattt acctaactta aacgaagaac aacgcaatgg tttcatccaa agcctaaaag
     1981 atgacccaag ccaaagcgct aaccttttag cagaagctaa aaagctaaat gatgctcaag
     2041 caccaaaagc tcaacaaaat gctttttatc aagtcttaaa tatgcctaac ttaaatgctg
     2101 atcaacgcaa tggttttatc caaagcctta aagatgatcc aagccaaagt gctaacgttt
     2161 taggtgaagc tcaaaaactt aatgactctc aagctccaaa agctgatgcg caacaaaata
     2221 acttcaacaa agatcaacaa agcgccttct atgaaatctt gaacatgcct aacttaaacg
     2281 aagcgcaacg taacggcttc attcaaagtc ttaaagacga cccaagccaa agcactaacg
     2341 ttttaggtga agctaaaaaa ttaaacgaat ctcaagcacc gaaagctgat aacaatttca
     2401 acaaagaaca acaaaatgct ttctatgaaa tcttgaatat gcctaactta aacgaagaac
     2461 aacgcaatgg tttcatccaa agcttaaaag atgacccaag ccaaagtgct aacctattgt
     2521 cagaagctaa aaagttaaat gaatctcaag caccgaaagc ggataacaaa ttcaacaaag
     2581 aacaacaaaa tgctttctat gaaatcttac atttacctaa cttaaacgaa gaacaacgca
     2641 atggtttcat ccaaagccta aaagatgacc caagccaaag cgctaacctt ttagcagaag
     2701 ctaaaaagct aaatgatgct caagcaccaa aataactcga gcaccaccac caccaccact
     2761 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     2821 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     2881 gaggaactat atccggatgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt
     2941 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3001 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3061 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3121 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3181 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3241 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3301 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3361 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     3421 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3481 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     3541 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3601 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3661 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3721 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     3781 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     3841 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3901 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3961 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     4021 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     4081 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4141 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4201 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4261 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4321 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     4381 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     4441 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4501 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4561 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4621 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4681 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4741 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4801 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     4861 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     4921 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     4981 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     5041 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     5101 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5161 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5221 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5281 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5341 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5401 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5461 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5521 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5581 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5641 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5701 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5761 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5821 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5881 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5941 ccatgcatta gttattaa
//
