LOCUS       sequence-134420-        4268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NLSPUFw_VP64 in expression vector pmax.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4268)
  AUTHORS   Cheng AW, Jillette N, Lee P, Plaskon D, Fujiwara Y, Wang W, 
            Taghbalout A, Wang H
  TITLE     Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation 
            and genomic labeling.
  JOURNAL   Cell Res. 2016 Feb;26(2):254-7. doi: 10.1038/cr.2016.3. Epub 2016 
            Jan 15.
  PUBMED    26768771
REFERENCE   2  (bases 1 to 4268)
  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/cr.2016.3";
            journalName: "Cell Res"; date: "2016-02"; volume: "26"; issue: "2"; 
            pages: "254-7"
COMMENT     Sequence Label: pAC1357-pmax-NLSPUFw_VP64
FEATURES             Location/Qualifiers
     source          1..4268
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          813..945
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     protein_bind    960..984
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1002..1011
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1023..1043
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2121..2270
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     protein_bind    complement(2298..2322)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2375..2416
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2442..2461)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    2471..2552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2496..2515
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(2750..3337)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2830..2849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3418..4212)
                     /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             complement(3418..4212)
                     /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(3529..3548)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4139..4158
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg
      721 tcgtttagtg aaccgtcaga tcactagtag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctc gactgatcac aggtaagtat caaggttaca agacaggttt
      841 aaggaggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg
      901 cacctattgg tcttactgac atccactttg cctttctctc cacagggaaa aaacaattga
      961 caagtttgta caaaaaagca ggctccgaat tcaccggtgc cgccaccatg gggatcctcc
     1021 cccccaagaa aaagaggaag gtatctagag gccgcagccg ccttttggaa gattttcgaa
     1081 acaaccggta ccccaattta caactgcggg agattgctgg acatataatg gaattttccc
     1141 aagaccagca tgggtccaga ttcattcagc tgaaactgga gcgtgccaca ccagctgagc
     1201 gccagcttgt cttcaatgaa atcctccagg ctgcctacca actcatggtg gatgtgtttg
     1261 gtaattacgt cattcagaag ttctttgaat ttggcagtct tgaacagaag ctggctttgg
     1321 cagaacggat tcgaggccac gtcctgtcat tggcactaca gatgtatggc tgccgtgtta
     1381 tccagaaagc tcttgagttt attccttcag accagcagaa tgagatggtt cgggaactag
     1441 atggccatgt cttgaagtgt gtgaaagatc agaatggcaa tcacgtggtt cagaaatgca
     1501 ttgaatgtgt acagccccag tctttgcaat ttatcatcga cgcgtttaag ggacaggtat
     1561 ttgccttatc cacacatcct tatggctgcc gagtgattca gagaatcctg gagcactgtc
     1621 tccctgacca gacactccct attttagagg agcttcacca gcacacagag cagctggtac
     1681 aggatcaata tggaaattat gtaatccaac atgtactgga gcacggtcgt cctgaggata
     1741 aaagcaaaat tgtagcagaa atccgaggca atgtacttgt attgagtcag cacaaatttg
     1801 caagcaatgt tgtggagaag tgtgttactc acgcctcacg tacggagcgc gctgtgctca
     1861 tcgacgaggt gtgcaccatg aacgacggtc cccacagtgc cttatacacc atgatgaagg
     1921 accagtatgc caactacgtg gtccagaaga tgattgacgt ggcggagcca ggccagcgga
     1981 agatcgtcat gcataagatc cggccccaca tcgcaactct tcgtaagtac acctatggca
     2041 agcacattct ggccaagctg gagaagtact acatgaagaa cggtgttgac ttaggggggc
     2101 cggccggatc cgggcgcgcc gacgcgctgg acgatttcga tctcgacatg ctgggttctg
     2161 atgccctcga tgactttgac ctggatatgt tgggaagcga cgcattggat gactttgatc
     2221 tggacatgct cggctccgat gctctggacg atttcgatct cgatatgtta tatatcgatt
     2281 gattaattaa gaattcgacc cagctttctt gtacaaagtg gttgatatcc agcacagtgg
     2341 cggccgctcg agtctagagg gcccgcggtt cgaaggtaag cctatcccta accctctcct
     2401 cggtctcgat tctacgcgta ccggttagca attgtttttt cgatgagttt ggacaaacca
     2461 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     2521 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     2581 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     2641 gtacttaaga gggggagacc aaagggcgag acgttaaggc ctcacgtgac atgtgagcaa
     2701 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2941 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3001 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3181 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3241 gagttggtag ctcttgatcc ggcaaacaaa ccacgctggt agcggtggtt tttttgtttg
     3301 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3361 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgccgtctca
     3421 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
     3481 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
     3541 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc
     3601 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac
     3661 gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag
     3721 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
     3781 tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
     3841 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga
     3901 tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt
     3961 gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc
     4021 tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
     4081 gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc
     4141 ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc
     4201 ttgttcaatc ataatattat tgaagcattt atcagggttc gtctcgtccc ggtctcctcc
     4261 catgcatg
//
