LOCUS       sequence-136520-        5012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BFPRKAB_PUFa in pCR8 gateway donor vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5012)
  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 5012)
  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: pAC1450_pCR8-BFPKRAB_PUFa
FEATURES             Location/Qualifiers
     source          1..5012
                     /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      686..695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             698..724
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             704..730
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             743..763
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             770..790
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             830..1525
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             1571..1756
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             1799..1819
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2891..2990)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3007..3026)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3008..3026)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3031..3047)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3031..3047)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3478..4269
                     /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      4362..4950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4851..4870
                     /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 catggcctat ccctatgacg tgcccgatta
      721 tgccagcctg ggcagcggct cccccaagaa aaaacgcaag gtggaagatc ctaagaaaaa
      781 gcggaaagtg gacggcattg gtagtgggag caacggcagc agcggatcca gcgagctgat
      841 taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc atcacttcaa
      901 gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt
      961 ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct tcctctacgg
     1021 cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc agtccttccc
     1081 tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac
     1141 ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa
     1201 cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccttcaccga
     1261 gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc tgaagctcgt
     1321 gggcgggagc catctgatcg caaacatcaa gaccacatat agatccaaga aacccgctaa
     1381 gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc
     1441 caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact gcgacctccc
     1501 tagcaaactg gggcacaagc ttaatggcgg tggcggaggg atggatgcta agtcactaac
     1561 tgcctggtcc cggacactgg tgaccttcaa ggatgtattt gtggacttca ccagggagga
     1621 gtggaagctg ctggacactg ctcagcagat cgtgtacaga aatgtgatgc tggagaacta
     1681 taagaacctg gtttccttgg gttatcagct tactaagcca gatgtgatcc tccggttgga
     1741 gaagggagaa gagcccggcg gttccggcgg agggtcgggg ccggccggga tcctcccccc
     1801 caagaaaaag aggaaggtat ctagaggccg cagccgcctt ttggaagatt ttcgaaacaa
     1861 ccggtacccc aatttacaac tgcgggagat tgctggacat ataatggaat tttcccaaga
     1921 ccagcatggg tccagattca ttcagctgaa actggagcgt gccacaccag ctgagcgcca
     1981 gcttgtcttc aatgaaatcc tccaggctgc ctaccaactc atggtggatg tgtttggtaa
     2041 ttacgtcatt cagaagttct ttgaatttgg cagtcttgaa cagaagctgg ctttggcaga
     2101 acggattcga ggccacgtcc tgtcattggc actacagatg tatggcagcc gtgttatcga
     2161 gaaagctctt gagtttattc cttcagacca gcagaatgag atggttcggg aactagatgg
     2221 ccatgtcttg aagtgtgtga aagatcagaa tggcaatcac gtggttcaga aatgcattga
     2281 atgtgtacag ccccagtctt tgcaatttat catcgatgcg tttaagggac aggtatttgc
     2341 cttatccaca catccttatg gctgccgagt gattcagaga atcctggagc actgtctccc
     2401 tgaccagaca ctccctattt tagaggagct tcaccagcac acagagcagc tggtacagga
     2461 tcaatatgga aattatgtaa tccaacatgt actggagcac ggtcgtcctg aggataaaag
     2521 caaaattgta gcagaaatcc gaggcaatgt acttgtattg agtcagcaca aatttgcaag
     2581 caatgttgtg gagaagtgtg ttactcacgc ctcacgtacg gagcgcgctg tgctcatcga
     2641 tgaggtgtgc accatgaacg acggtcccca cagtgcctta tacaccatga tgaaggacca
     2701 gtatgccaac tacgtggtcc agaagatgat tgacgtggcg gagccaggcc agcggaagat
     2761 cgtcatgcat aagatccggc cccacatcgc aactcttcgt aagtacacct atggcaagca
     2821 cattctggcc aagctggaga agtactacat gaagaacggt gttgacttag ggtgattaat
     2881 taagaattcg acccagcttt cttgtacaaa gttggcatta taaaaaataa ttgctcatca
     2941 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     3001 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     3061 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgcctcctc
     3121 tagaccagcc aggacagaaa tgcctcgact tcgctgctgc ccaaggttgc cgggtgacgc
     3181 acaccgtgga aacggatgaa ggcacgaacc cagtggacat aagcctgttc ggttcgtaag
     3241 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
     3301 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag
     3361 tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta
     3421 tggagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacatcatg
     3481 agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag
     3541 cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc
     3601 ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca
     3661 acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag
     3721 attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat
     3781 ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc
     3841 ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat
     3901 agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat
     3961 ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc
     4021 gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa
     4081 atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag
     4141 cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg
     4201 cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc
     4261 ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta cgcgtcgttc
     4321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4501 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4741 ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4981 ctggcctttt gctggccttt tgctcacatg tt
//
