LOCUS       sequence-134385-        6491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4xNLS_PUFa_2xNLS_mCBPHAT in pCR8 Gateway donor vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6491)
  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 6491)
  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: pAC1415-pCR8-4xNLS_PUFa_2xNLS_mCBPHAT
FEATURES             Location/Qualifiers
     source          1..6491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             719..739
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             743..763
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             767..787
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             878..898
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1955..1975
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1979..1999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      3680..3689
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(4370..4469)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4486..4505)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4487..4505)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4510..4526)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4510..4526)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4957..5748
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      5841..6429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6330..6349
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gaattcaccg gtgccgccac catgatcgat ggcggaggcg gatctgatcc
      721 aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag gtagatccaa aaaagaagag
      781 aaaggtagga tctaccggat ctagaaacga tggtggtggt ggaagcgggg gtgggggcag
      841 cggtggaggg ggaagcgggc gcgccgggat cctccccccc aagaaaaaga ggaaggtatc
      901 tagaggccgc agccgccttt tggaagattt tcgaaacaac cggtacccca atttacaact
      961 gcgggagatt gctggacata taatggaatt ttcccaagac cagcatgggt ccagattcat
     1021 tcagctgaaa ctggagcgtg ccacaccagc tgagcgccag cttgtcttca atgaaatcct
     1081 ccaggctgcc taccaactca tggtggatgt gtttggtaat tacgtcattc agaagttctt
     1141 tgaatttggc agtcttgaac agaagctggc tttggcagaa cggattcgag gccacgtcct
     1201 gtcattggca ctacagatgt atggcagccg tgttatcgag aaagctcttg agtttattcc
     1261 ttcagaccag cagaatgaga tggttcggga actagatggc catgtcttga agtgtgtgaa
     1321 agatcagaat ggcaatcacg tggttcagaa atgcattgaa tgtgtacagc cccagtcttt
     1381 gcaatttatc atcgacgcgt ttaagggaca ggtatttgcc ttatccacac atccttatgg
     1441 ctgccgagtg attcagagaa tcctggagca ctgtctccct gaccagacac tccctatttt
     1501 agaggagctt caccagcaca cagagcagct ggtacaggat caatatggaa attatgtaat
     1561 ccaacatgta ctggagcacg gtcgtcctga ggataaaagc aaaattgtag cagaaatccg
     1621 aggcaatgta cttgtattga gtcagcacaa atttgcaagc aatgttgtgg agaagtgtgt
     1681 tactcacgcc tcacgtacgg agcgcgctgt gctcatcgac gaggtgtgca ccatgaacga
     1741 cggtccccac agtgccttat acaccatgat gaaggaccag tatgccaact acgtggtcca
     1801 gaagatgatt gacgtggcgg agccaggcca gcggaagatc gtcatgcata agatccggcc
     1861 ccacatcgca actcttcgta agtacaccta tggcaagcac attctggcca agctggagaa
     1921 gtactacatg aagaacggtg ttgacttagg ggacccaaag aagaagcgca aagtggatcc
     1981 taaaaagaaa agaaaggtag gcggccgcgg gggaggcggt tccggtggcg gcggaagcgg
     2041 aggtggagga tcagggccgg ccatctttaa acccgaggag ctacgccagg cacttatgcc
     2101 aactctagaa gcactctatc gacaggaccc agagtctttg ccttttcgtc agcctgtaga
     2161 tcctcagctc ctaggaatcc cagattattt tgatatagtg aagaatccta tggacctttc
     2221 taccatcaaa cgaaagctgg acacagggca atatcaagaa ccctggcagt atgtggatga
     2281 tgtctggctt atgttcaaca atgcgtggct atataatcgt aaaacgtccc gtgtatataa
     2341 attttgcagt aaacttgcag aggtctttga acaagaaatt gaccctgtca tgcagtctct
     2401 tggatattgc tgtggacgaa agtatgagtt ctccccacag actttgtgct gttacggaaa
     2461 gcagctgtgt acaattcctc gtgatgcagc ctactacagc tatcagaata ggtatcattt
     2521 ctgtgagaag tgtttcacag agatccaggg cgagaatgtg accctgggtg acgacccttc
     2581 ccaacctcag acgacaattt ccaaggatca atttgaaaag aagaaaaatg ataccttaga
     2641 tcctgaacct tttgttgact gcaaagagtg tggccggaag atgcatcaga tttgtgttct
     2701 acactatgac atcatttggc cttcaggttt tgtgtgtgac aactgtttga agaaaactgg
     2761 cagacctcgg aaagaaaaca aattcagtgc taagaggctg cagaccacac gattgggaaa
     2821 ccacttagaa gacagagtga ataagttttt gcggcgccag aatcaccctg aagctgggga
     2881 ggtttttgtc agagtggtgg ccagctcaga caagactgtg gaggtcaagc cgggaatgaa
     2941 gtcaaggttt gtggattctg gagagatgtc ggaatctttc ccatatcgta ccaaagcact
     3001 ctttgctttt gaggagatcg atggagtcga tgtgtgcttt tttgggatgc atgtgcaaga
     3061 atacggctct gattgccccc caccaaatac aaggcgtgta tacatatctt atctggacag
     3121 tattcatttc ttccggcccc gctgcctccg gacagctgtt taccatgaga tcctcatcgg
     3181 atatctcgag tatgtgaaga aattggggta tgtgacagga catatttggg cctgtccccc
     3241 aagtgaagga gatgactata tctttcattg ccacccccct gaccagaaaa tccccaaacc
     3301 aaaacgacta caggagtggt acaagaagat gctggacaag gcgtttgcag agaggatcat
     3361 taacgactat aaggacatct tcaaacaagc gaacgaagac aggctcacga gtgccaagga
     3421 gttgccctat tttgaaggag atttctggcc taatgtgttg gaagaaagca ttaaggaact
     3481 agaacaagaa gaagaagaaa ggaaaaaaga agagagtact gcagcgagtg agactcctga
     3541 gggcagtcag ggtgacagca aaaatgcgaa gaaaaagaac aacaagaaga ccaacaaaaa
     3601 caaaagcagc attagccgcg ccaacaagaa gaagcccagc atgcccaatg tttccaacga
     3661 cctgtcgcag aagctgtatg ccaccatgga gaagcacaag gaggtattct ttgtgattca
     3721 tctgcatgct gggcctgtta tcagcactca gccccccatc gtggaccctg atcctctgct
     3781 tagctgtgac ctcatggatg ggcgagatgc cttcctcacc ctggccagag acaagcactg
     3841 ggaattctct tccttacgcc gctccaaatg gtccactctg tgcatgctgg tggagctgca
     3901 cacacagggc caggaccgct ttgtttatac ctgcaatgag tgcaaacacc atgtggaaac
     3961 acgctggcac tgcactgtgt gtgaggacta tgacctttgt atcaattgct acaacacaaa
     4021 gagccacacc cataagatgg tgaagtgggg gctaggccta gatgatgagg gcagcagtca
     4081 gggtgagcca cagtccaaga gcccccagga atcccggcgt ctcagcatcc agcgctgcat
     4141 ccagtccctg gtgcatgcct gccagtgtcg caatgccaac tgctcactgc cgtcttgcca
     4201 gaagatgaag cgagtcgtgc agcacaccaa gggctgcaag cgcaagacta atggaggatg
     4261 cccagtgtgc aagcagctca ttgctctttg ctgctaccac gccaaacact gccaagaaaa
     4321 taaatgccct gtgcccttct gcctcaacat ctagttaatt aagaattcga cccagctttc
     4381 ttgtacaaag ttggcattat aaaaaataat tgctcatcaa tttgttgcaa cgaacaggtc
     4441 actatcagtc aaaataaaat cattatttgc catccagctg atatccccta tagtgagtcg
     4501 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt
     4561 tacattgcac aagataaaaa tatatcatca tgcctcctct agaccagcca ggacagaaat
     4621 gcctcgactt cgctgctgcc caaggttgcc gggtgacgca caccgtggaa acggatgaag
     4681 gcacgaaccc agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg
     4741 cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg
     4801 gttttcatgg cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag
     4861 cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg
     4921 cagcagggca gtcgccctaa aacaaagtta aacatcatga gggaagcggt gatcgccgaa
     4981 gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg
     5041 ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt
     5101 gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac
     5161 gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc
     5221 accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa
     5281 tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac
     5341 attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca
     5401 gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa
     5461 accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt
     5521 acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct
     5581 gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga
     5641 caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa
     5701 tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc ctcgagccac
     5761 ccatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta
     5821 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5881 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5941 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     6001 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6061 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6121 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6181 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa
     6241 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6301 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6361 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6421 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6481 gctcacatgt t
//
