LOCUS       sequence-134384-        6491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mCBPHAT_4xNLS_PUFa_2xNLS 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: pAC1416-pCR8-mCBPHAT_4xNLS_PUFa_2xNLS
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"
     regulatory      2312..2321
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3008..3028
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3032..3052
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3056..3076
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3167..3187
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4244..4264
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4268..4288
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     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 catgatcttt aaacccgagg agctacgcca
      721 ggcacttatg ccaactctag aagcactcta tcgacaggac ccagagtctt tgccttttcg
      781 tcagcctgta gatcctcagc tcctaggaat cccagattat tttgatatag tgaagaatcc
      841 tatggacctt tctaccatca aacgaaagct ggacacaggg caatatcaag aaccctggca
      901 gtatgtggat gatgtctggc ttatgttcaa caatgcgtgg ctatataatc gtaaaacgtc
      961 ccgtgtatat aaattttgca gtaaacttgc agaggtcttt gaacaagaaa ttgaccctgt
     1021 catgcagtct cttggatatt gctgtggacg aaagtatgag ttctccccac agactttgtg
     1081 ctgttacgga aagcagctgt gtacaattcc tcgtgatgca gcctactaca gctatcagaa
     1141 taggtatcat ttctgtgaga agtgtttcac agagatccag ggcgagaatg tgaccctggg
     1201 tgacgaccct tcccaacctc agacgacaat ttccaaggat caatttgaaa agaagaaaaa
     1261 tgatacctta gatcctgaac cttttgttga ctgcaaagag tgtggccgga agatgcatca
     1321 gatttgtgtt ctacactatg acatcatttg gccttcaggt tttgtgtgtg acaactgttt
     1381 gaagaaaact ggcagacctc ggaaagaaaa caaattcagt gctaagaggc tgcagaccac
     1441 acgattggga aaccacttag aagacagagt gaataagttt ttgcggcgcc agaatcaccc
     1501 tgaagctggg gaggtttttg tcagagtggt ggccagctca gacaagactg tggaggtcaa
     1561 gccgggaatg aagtcaaggt ttgtggattc tggagagatg tcggaatctt tcccatatcg
     1621 taccaaagca ctctttgctt ttgaggagat cgatggagtc gatgtgtgct tttttgggat
     1681 gcatgtgcaa gaatacggct ctgattgccc cccaccaaat acaaggcgtg tatacatatc
     1741 ttatctggac agtattcatt tcttccggcc ccgctgcctc cggacagctg tttaccatga
     1801 gatcctcatc ggatatctcg agtatgtgaa gaaattgggg tatgtgacag gacatatttg
     1861 ggcctgtccc ccaagtgaag gagatgacta tatctttcat tgccaccccc ctgaccagaa
     1921 aatccccaaa ccaaaacgac tacaggagtg gtacaagaag atgctggaca aggcgtttgc
     1981 agagaggatc attaacgact ataaggacat cttcaaacaa gcgaacgaag acaggctcac
     2041 gagtgccaag gagttgccct attttgaagg agatttctgg cctaatgtgt tggaagaaag
     2101 cattaaggaa ctagaacaag aagaagaaga aaggaaaaaa gaagagagta ctgcagcgag
     2161 tgagactcct gagggcagtc agggtgacag caaaaatgcg aagaaaaaga acaacaagaa
     2221 gaccaacaaa aacaaaagca gcattagccg cgccaacaag aagaagccca gcatgcccaa
     2281 tgtttccaac gacctgtcgc agaagctgta tgccaccatg gagaagcaca aggaggtatt
     2341 ctttgtgatt catctgcatg ctgggcctgt tatcagcact cagcccccca tcgtggaccc
     2401 tgatcctctg cttagctgtg acctcatgga tgggcgagat gccttcctca ccctggccag
     2461 agacaagcac tgggaattct cttccttacg ccgctccaaa tggtccactc tgtgcatgct
     2521 ggtggagctg cacacacagg gccaggaccg ctttgtttat acctgcaatg agtgcaaaca
     2581 ccatgtggaa acacgctggc actgcactgt gtgtgaggac tatgaccttt gtatcaattg
     2641 ctacaacaca aagagccaca cccataagat ggtgaagtgg gggctaggcc tagatgatga
     2701 gggcagcagt cagggtgagc cacagtccaa gagcccccag gaatcccggc gtctcagcat
     2761 ccagcgctgc atccagtccc tggtgcatgc ctgccagtgt cgcaatgcca actgctcact
     2821 gccgtcttgc cagaagatga agcgagtcgt gcagcacacc aagggctgca agcgcaagac
     2881 taatggagga tgcccagtgt gcaagcagct cattgctctt tgctgctacc acgccaaaca
     2941 ctgccaagaa aataaatgcc ctgtgccctt ctgcctcaac atcaacgatg gcggaggcgg
     3001 atctgatcca aaaaagaaga gaaaggtaga tccaaaaaag aagagaaagg tagatccaaa
     3061 aaagaagaga aaggtaggat ctaccggatc tagaaacgat ggtggtggtg gaagcggggg
     3121 tgggggcagc ggtggagggg gaagcgggcg cgccgggatc ctccccccca agaaaaagag
     3181 gaaggtatct agaggccgca gccgcctttt ggaagatttt cgaaacaacc ggtaccccaa
     3241 tttacaactg cgggagattg ctggacatat aatggaattt tcccaagacc agcatgggtc
     3301 cagattcatt cagctgaaac tggagcgtgc cacaccagct gagcgccagc ttgtcttcaa
     3361 tgaaatcctc caggctgcct accaactcat ggtggatgtg tttggtaatt acgtcattca
     3421 gaagttcttt gaatttggca gtcttgaaca gaagctggct ttggcagaac ggattcgagg
     3481 ccacgtcctg tcattggcac tacagatgta tggcagccgt gttatcgaga aagctcttga
     3541 gtttattcct tcagaccagc agaatgagat ggttcgggaa ctagatggcc atgtcttgaa
     3601 gtgtgtgaaa gatcagaatg gcaatcacgt ggttcagaaa tgcattgaat gtgtacagcc
     3661 ccagtctttg caatttatca tcgacgcgtt taagggacag gtatttgcct tatccacaca
     3721 tccttatggc tgccgagtga ttcagagaat cctggagcac tgtctccctg accagacact
     3781 ccctatttta gaggagcttc accagcacac agagcagctg gtacaggatc aatatggaaa
     3841 ttatgtaatc caacatgtac tggagcacgg tcgtcctgag gataaaagca aaattgtagc
     3901 agaaatccga ggcaatgtac ttgtattgag tcagcacaaa tttgcaagca atgttgtgga
     3961 gaagtgtgtt actcacgcct cacgtacgga gcgcgctgtg ctcatcgacg aggtgtgcac
     4021 catgaacgac ggtccccaca gtgccttata caccatgatg aaggaccagt atgccaacta
     4081 cgtggtccag aagatgattg acgtggcgga gccaggccag cggaagatcg tcatgcataa
     4141 gatccggccc cacatcgcaa ctcttcgtaa gtacacctat ggcaagcaca ttctggccaa
     4201 gctggagaag tactacatga agaacggtgt tgacttaggg gacccaaaga agaagcgcaa
     4261 agtggatcct aaaaagaaaa gaaaggtagg cggccgcggg ggaggcggtt ccggtggcgg
     4321 cggaagcgga ggtggaggat cagggccggc ctgattaatt 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
//
