LOCUS       sequence-136967-        4202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NLSPUFc pCR8 gateway donor vector for insertion of N-terminal domain
            (SgrAI or AgeI with ClaI) and C-terminal domain (FseI PacI). Grow in
            dcm- cells (e.g., NEB C3040) to prepare vector for ClaI digest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4202)
  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 4202)
  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: pAC1408-pCR8-4xNLS_PUFc_2xNLS
FEATURES             Location/Qualifiers
     source          1..4202
                     /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"
     protein_bind    complement(2081..2180)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2197..2216)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2198..2216)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2221..2237)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2221..2237)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2668..3459
                     /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      3552..4140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4041..4060
                     /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 gttatgtaat
     1561 cgaacatgta ctggagcacg gtcgtcctga ggataaaagc aaaattgtag cagaaatccg
     1621 aggcaatgta cttgtattga gtcagcacaa atttgcaaac aatgttgtgc 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 cctgattaat taagaattcg acccagcttt cttgtacaaa
     2101 gttggcatta taaaaaataa ttgctcatca atttgttgca acgaacaggt cactatcagt
     2161 caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg
     2221 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca
     2281 caagataaaa atatatcatc atgcctcctc tagaccagcc aggacagaaa tgcctcgact
     2341 tcgctgctgc ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc
     2401 cagtggacat aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat gcgctcacgc
     2461 aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg
     2521 gcttgttatg actgtttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg
     2581 cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc
     2641 agtcgcccta aaacaaagtt aaacatcatg agggaagcgg tgatcgccga agtatcgact
     2701 caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta
     2761 catttgtacg gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg
     2821 gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg
     2881 gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt
     2941 gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa
     3001 tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg
     3061 gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag
     3121 gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg
     3181 ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc
     3241 cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg
     3301 gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat
     3361 cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccac
     3421 tacgtgaaag gcgagatcac caaggtagtc ggcaaataac cctcgagcca cccatgacca
     3481 aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc
     3541 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3601 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3661 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3721 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3781 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3841 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3901 gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg
     3961 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4021 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4081 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4141 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4201 tt
//
