LOCUS       sequence-134399-        4694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Clover_NLSPUFa in pCR8 gateway donor vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4694)
  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 4694)
  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: pAC1402-pCR8-Clover_NLSPUFa
FEATURES             Location/Qualifiers
     source          1..4694
                     /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             join(692..694,695..697,698..1408)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(737..758)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1345..1366
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1424..1442)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1425..1442)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1481..1501
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2573..2672)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2689..2708)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2690..2708)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2713..2729)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2713..2729)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3160..3951
                     /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      4044..4632
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4533..4552
                     /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 catggtgagc aagggcgagg agctgttcac
      721 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      781 ccgcggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac
      841 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcgtggc
      901 ctgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      961 cgaaggctac gtccaggagc gcaccatctc tttcaaggac gacggtacct acaagacccg
     1021 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1081 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca acagccacaa
     1141 cgtctatatc acggccgaca agcagaagaa cggcatcaag gctaacttca agatccgcca
     1201 caacgttgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1261 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc catcagtccg ccctgagcaa
     1321 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1381 tacacatggc atggacgagc tgtacaagtc tagagggccc tattctatag tgtcacctaa
     1441 atgcggaggc ggcggtagcg ggccggccgg gatcctcccc cccaagaaaa agaggaaggt
     1501 atctagaggc cgcagccgcc ttttggaaga ttttcgaaac aaccggtacc ccaatttaca
     1561 actgcgggag attgctggac atataatgga attttcccaa gaccagcatg ggtccagatt
     1621 cattcagctg aaactggagc gtgccacacc agctgagcgc cagcttgtct tcaatgaaat
     1681 cctccaggct gcctaccaac tcatggtgga tgtgtttggt aattacgtca ttcagaagtt
     1741 ctttgaattt ggcagtcttg aacagaagct ggctttggca gaacggattc gaggccacgt
     1801 cctgtcattg gcactacaga tgtatggcag ccgtgttatc gagaaagctc ttgagtttat
     1861 tccttcagac cagcagaatg agatggttcg ggaactagat ggccatgtct tgaagtgtgt
     1921 gaaagatcag aatggcaatc acgtggttca gaaatgcatt gaatgtgtac agccccagtc
     1981 tttgcaattt atcatcgatg cgtttaaggg acaggtattt gccttatcca cacatcctta
     2041 tggctgccga gtgattcaga gaatcctgga gcactgtctc cctgaccaga cactccctat
     2101 tttagaggag cttcaccagc acacagagca gctggtacag gatcaatatg gaaattatgt
     2161 aatccaacat gtactggagc acggtcgtcc tgaggataaa agcaaaattg tagcagaaat
     2221 ccgaggcaat gtacttgtat tgagtcagca caaatttgca agcaatgttg tggagaagtg
     2281 tgttactcac gcctcacgta cggagcgcgc tgtgctcatc gatgaggtgt gcaccatgaa
     2341 cgacggtccc cacagtgcct tatacaccat gatgaaggac cagtatgcca actacgtggt
     2401 ccagaagatg attgacgtgg cggagccagg ccagcggaag atcgtcatgc ataagatccg
     2461 gccccacatc gcaactcttc gtaagtacac ctatggcaag cacattctgg ccaagctgga
     2521 gaagtactac atgaagaacg gtgttgactt agggtgatta attaagaatt cgacccagct
     2581 ttcttgtaca aagttggcat tataaaaaat aattgctcat caatttgttg caacgaacag
     2641 gtcactatca gtcaaaataa aatcattatt tgccatccag ctgatatccc ctatagtgag
     2701 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
     2761 tgttacattg cacaagataa aaatatatca tcatgcctcc tctagaccag ccaggacaga
     2821 aatgcctcga cttcgctgct gcccaaggtt gccgggtgac gcacaccgtg gaaacggatg
     2881 aaggcacgaa cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt
     2941 atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg
     3001 gcggttttca tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc
     3061 aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt
     3121 acgcagcagg gcagtcgccc taaaacaaag ttaaacatca tgagggaagc ggtgatcgcc
     3181 gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg
     3241 ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat
     3301 attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc
     3361 aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa
     3421 gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg
     3481 caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc
     3541 gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt
     3601 ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat
     3661 gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg
     3721 cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc
     3781 gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct
     3841 agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa
     3901 gaatttgtcc actacgtgaa aggcgagatc accaaggtag tcggcaaata accctcgagc
     3961 cacccatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc
     4021 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4081 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4141 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     4201 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4261 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4321 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4381 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagcattga
     4441 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4501 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4561 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4621 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4681 tttgctcaca tgtt
//
