LOCUS       sequence-457131-        4754 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes 3xFLAG-NLS/SV40-AcGFP-NES/PKI which is a AcGFP with an 
            N-terminal SV40 NLS and a C-terminal PKI NES that shuttles between 
            the nucleus and the cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4754)
  AUTHORS   Vercruysse T, Vanstreels E, Jacquemyn M, Boland S, Kilonda A, 
            Allasia S, Vandecaetsbeek I, Klaassen H, Versele M, Chaltin P, 
            Marchand A, Daelemans D
  TITLE     Ibetazol, a novel inhibitor of importin beta1-mediated nuclear 
            import.
  JOURNAL   Commun Biol. 2024 Nov 23;7(1):1560. doi: 10.1038/s42003-024-07237-8.
  PUBMED    39580542
REFERENCE   2  (bases 1 to 4754)
  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: "Commun 
            Biol."; date: "2024-11-23"; volume: "7(1):1560. doi"; pages: " 
            10.1038/s42003-024-07237-8"
COMMENT     Sequence Label: pGL4-CMV-3xFLAG-NLS/SV40-AcGFP-NES/PKI-P2A-Puro
FEATURES             Location/Qualifiers
     source          1..4754
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..49
                     /note="synthetic polyadenylation signal"
     misc_feature    63..154
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     103..122
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     enhancer        221..600
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        601..804
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     755..775
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     849..868
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        849..867
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             912..977
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             996..1016
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1017..1730
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="VSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDDG
                     NYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFG
                     FVTAAAITHGMDELYK"
     CDS             1782..1838
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     polyA_signal    2462..2583
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2473..2492)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2527..2546
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2831..2848)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3002..3590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3082..3101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3790..4581,4582..4650))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
       61 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
      121 cccagtgcaa gtgcaggtgc cagaacattt ctcttaatga tagctagcgg gccagtaccc
      181 aaaaagcggg gggtctggtg gcagtggagg gggatccgtt gacattgatt attgactagt
      241 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      301 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      361 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      421 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      481 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      541 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      601 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      661 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      721 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      781 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      841 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc
      901 ttggtaccat ggactacaaa gaccatgacg gtgattataa agatcatgac atcgattaca
      961 aggatgacga tgacaagctt gcggccgcga attcaccaaa aaagaagaga aaggtagtga
     1021 gcaagggcgc cgagctgttc accggcatcg tgcccatcct gatcgagctg aatggcgatg
     1081 tgaatggcca caagttcagc gtgagcggcg agggcgaggg cgatgccacc tacggcaagc
     1141 tgaccctgaa gttcatctgc accaccggca agctgcctgt gccctggccc accctggtga
     1201 ccaccctgag ctacggcgtg cagtgcttct cacgctaccc cgatcacatg aagcagcacg
     1261 acttcttcaa gagcgccatg cctgagggct acatccagga gcgcaccatc ttcttcgagg
     1321 atgacggcaa ctacaagtcg cgcgccgagg tgaagttcga gggcgatacc ctggtgaatc
     1381 gcatcgagct gaccggcacc gatttcaagg aggatggcaa catcctgggc aataagatgg
     1441 agtacaacta caacgcccac aatgtgtaca tcatgaccga caaggccaag aatggcatca
     1501 aggtgaactt caagatccgc cacaacatcg aggatggcag cgtgcagctg gccgaccact
     1561 accagcagaa tacccccatc ggcgatggcc ctgtgctgct gcccgataac cactacctgt
     1621 ccacccagag cgccctgtcc aaggacccca acgagaagcg cgatcacatg atctacttcg
     1681 gcttcgtgac cgccgccgcc atcacccacg gcatggatga gctgtacaag gcaccggccg
     1741 gattagcctt gaaattagca ggtcttgata tcggtagtgg tgccacgaat tttagtctcc
     1801 tcaagcaagc tggggacgtc gaggagaatc ctggcccaat gactgaatac aagcctactg
     1861 tcaggttggc tacaagagac gacgttccta gagccgtgag aactctggct gcagccttcg
     1921 ccgactaccc cgccacgaga cacaccgttg acccagatcg gcatattgag agagtgactg
     1981 aactgcagga gctgtttctt acaagagttg gcctcgacat aggcaaggtg tgggtggcgg
     2041 acgacggcgc cgccgtggcc gtctggacca ctcccgaatc agttgaggct ggcgccgtat
     2101 tcgctgagat cggcccgaga atggctgagc tcagcgggag taggctcgcg gcacagcagc
     2161 aaatggaggg actgctggca ccacacaggc ccaaagaacc cgcctggttc ctggcaaccg
     2221 tcggtgtatc tcccgatcat caggggaaag gtctgggctc tgccgtagtg ctccctggcg
     2281 tggaggcagc tgagagagca ggagtacctg ccttcttgga gacctccgct ccaaggaatc
     2341 ttcccttcta tgaacggttg ggcttcaccg tgacagccga cgtggaagtc cccgaaggcc
     2401 cccgcacttg gtgcatgacg aggaagcctg gagcgtgagg gcccgcttcg agcagacatg
     2461 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     2521 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     2581 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     2641 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaagatcc gtcgaccgat
     2701 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     2761 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     2821 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2881 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2941 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3001 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3061 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3121 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3181 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3241 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3301 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3361 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3421 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3481 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3541 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3601 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3661 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3721 aatcaatcta aagtatatat gagtaaactt ggtctgacag cggccgcaaa tgctaaacca
     3781 ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct gcctatttcg
     3841 ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg agggcttacc
     3901 atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc ccgatttgtc
     3961 agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta ctttgtccgc
     4021 ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc cagtgagtag
     4081 tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt cgttcggtat
     4141 ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac ccatattatg
     4201 aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt tggccgcggt
     4261 gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc catccgtaag
     4321 atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt gtatacggcg
     4381 accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata gcagtacttt
     4441 gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga tcttgccgct
     4501 attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag catcttttac
     4561 tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa agaagggaat
     4621 gagtgcgaca cgaaaatgtt ggatgctcat actcgtcctt tttcaatatt attgaagcat
     4681 ttatcagggt tactagtacg tctctcaagg ataagtaagt aatattaagg tacgggaggt
     4741 attggacagg ccgc
//
