LOCUS       sequence-457259-        7678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes NES/PKI-mNeonGreen2x-NLS/FUS which is a tandem of mNeongreen
            with an N-terminal PKI NES and a C-terminal FUS NLS that shuttles 
            between the nucleus and the cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7678)
  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 7678)
  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: pcDNA3.1-CMV-Puro-P2A-NES/PKI-mNeonGreen2x-NLS...
FEATURES             Location/Qualifiers
     source          1..7678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1503..1559
                     /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"
     CDS             1605..2306
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEEL
                     NLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
                     SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTF
                     KWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA
                     FTDVMGMDELYK"
     regulatory      1854..1863
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2334..3035
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEEL
                     NLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
                     SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTF
                     KWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA
                     FTDVMGMDELYK"
     regulatory      2583..2592
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3269..3286)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3275..3499
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3545..3973
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3632..3651)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3842..3863
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3982..4002)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3987..4316
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4167..4302
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4229..4248
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4383..5177
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4437..4456)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5047..5066
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5351..5472
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5388..5407)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5442..5461
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5521..5537)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5521..5537)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5534..5556)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5545..5561
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5569..5575,5576..5593,5594..5599))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5614..5635
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5752..5769)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5923..6511)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6003..6022)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6682..7473,7474..7542))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7305..7324
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7543..7647)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggcatga
      901 ccgagtacaa gcctactgtc aggttggcta caagagacga cgttcctaga gccgtgagaa
      961 ctctggctgc agccttcgcc gactaccccg ccacgagaca caccgttgac ccagatcggc
     1021 atattgagag agtgactgaa ctgcaggagc tgtttcttac aagagttggc ctcgacatag
     1081 gcaaggtgtg ggtggcggac gacggcgccg ccgtggccgt ctggaccact cccgaatcag
     1141 ttgaggctgg cgccgtattc gctgagatcg gcccgagaat ggctgagctc agcgggagta
     1201 ggctcgcggc acagcagcaa atggagggac tgctggcacc acacaggccc aaagaacccg
     1261 cctggttcct ggcaaccgtc ggtgtatctc ccgatcatca ggggaaaggt ctgggctctg
     1321 ccgtagtgct ccctggcgtg gaggcagctg agagagcagg agtacctgcc ttcttggaga
     1381 cctccgctcc aaggaatctt cccttctatg aacggttggg cttcaccgtg acagccgacg
     1441 tggaagtccc cgaaggcccc cgcacttggt gcatgacgag gaagcctgga gcggggagtg
     1501 gcgcaaccaa ttttagtctc ctcaaacagg ccggcgatgt cgaagaaaat ccgggcccta
     1561 tgagccttaa cgaactggcc ctgccgctgg ctggtttgga catcgtgagc aagggcgagg
     1621 aggataacgc ctctctccca gcgacacatg agttacacat ctttggctcc atcaacggtg
     1681 tggactttga catggtgggt cagggcaccg gcaatccaaa tgatggttat gaggagttaa
     1741 acctgaagtc caccaagggt gacctccagt tctccccctg gattctggtc cctcatatcg
     1801 ggtatggctt ccatcagtac ctgccctacc ctgacgggat gtcgcctttc caggccgcca
     1861 tggtagatgg cagcggatac caagtccatc gcacaatgca gtttgaagat ggtgcctccc
     1921 ttactgttaa ctaccgctac acctacgagg gaagccacat caaaggagag gcccaggtga
     1981 aggggactgg tttccctgct gacggtcctg tgatgaccaa ctcgctgacc gctgcggact
     2041 ggtgcaggtc gaagaagact taccccaacg acaaaaccat catcagtacc tttaagtgga
     2101 gttacaccac tggaaatggc aagcgctacc ggagcactgc gcggaccacc tacacctttg
     2161 ccaagccaat ggcggctaac tatctgaaga accagccgat gtacgtgttc cgtaagacgg
     2221 agctcaagca ctccaagacc gagctcaact tcaaggagtg gcaaaaggcc tttaccgatg
     2281 tgatgggcat ggacgagctg tacaaggtcg gcgcaggcgc actcgaggga tccgtgagca
     2341 agggcgagga ggataacgcc tctctcccag cgacacatga gttacacatc tttggctcca
     2401 tcaacggtgt ggactttgac atggtgggtc agggcaccgg caatccaaat gatggttatg
     2461 aggagttaaa cctgaagtcc accaagggtg acctccagtt ctccccctgg attctggtcc
     2521 ctcatatcgg gtatggcttc catcagtacc tgccctaccc tgacgggatg tcgcctttcc
     2581 aggccgccat ggtagatggc agcggatacc aagtccatcg cacaatgcag tttgaagatg
     2641 gtgcctccct tactgttaac taccgctaca cctacgaggg aagccacatc aaaggagagg
     2701 cccaggtgaa ggggactggt ttccctgctg acggtcctgt gatgaccaac tcgctgaccg
     2761 ctgcggactg gtgcaggtcg aagaagactt accccaacga caaaaccatc atcagtacct
     2821 ttaagtggag ttacaccact ggaaatggca agcgctaccg gagcactgcg cggaccacct
     2881 acacctttgc caagccaatg gcggctaact atctgaagaa ccagccgatg tacgtgttcc
     2941 gtaagacgga gctcaagcac tccaagaccg agctcaactt caaggagtgg caaaaggcct
     3001 ttaccgatgt gatgggcatg gacgagctgt acaagggcag tggcagtcgc ggcggccgcg
     3061 gcggcggcga tcgcggcggc tttggcccgg gcaaaatgga tagccgcggc gaacatcgcc
     3121 aggatcgccg cgaacgcccg tactagcgtt taaacttaag cttggtaccg agctcggatc
     3181 cactagtcca gtgtggtgga attctgcaga tatccagcac agtggcggcc gctcgagtct
     3241 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     3301 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     3361 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     3421 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     3481 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     3541 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3601 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3661 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3721 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3781 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3841 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3901 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3961 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     4021 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     4081 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     4141 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4201 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     4261 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     4321 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     4381 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     4441 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     4501 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     4561 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     4621 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     4681 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     4741 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     4801 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     4861 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     4921 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     4981 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     5041 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     5101 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     5161 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     5221 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5281 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5341 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5401 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5461 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     5521 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5581 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5641 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5701 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5761 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5821 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5881 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5941 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6001 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6061 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6121 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6181 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6241 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6301 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6361 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6421 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6481 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6541 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6601 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6661 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6721 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6781 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6841 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6901 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6961 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7021 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7081 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7141 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7201 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7261 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7321 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7381 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7441 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7501 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7561 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7621 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
