LOCUS       sequence-136519-        4364 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  KRAB_PUFa in pmax-DEST expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4364)
  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 4364)
  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: pAC1449_pmax-KRAB_PUFa
FEATURES             Location/Qualifiers
     source          1..4364
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          813..945
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     protein_bind    960..984
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1002..1011
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1017..1037
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1077..1271
                     /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
                     QLTKPDVILRLEKGEEPWLV"
     CDS             1302..1322
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2394..2418)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2471..2512
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2538..2557)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    2567..2648
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2592..2611
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(2846..3433)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2926..2945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3514..4308)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3514..4308)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3625..3644)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4235..4254
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg
      721 tcgtttagtg aaccgtcaga tcactagtag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctc gactgatcac aggtaagtat caaggttaca agacaggttt
      841 aaggaggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg
      901 cacctattgg tcttactgac atccactttg cctttctctc cacagggaaa aaacaattga
      961 caagtttgta caaaaaagca ggctccgaat tcaccggtgc cgccaccatg gggagcccca
     1021 agaagaagag aaaggtggag gccagcatgg acgcgaaatc acttacggca tggtcgagaa
     1081 cactggttac gttcaaggac gtgtttgtgg actttacacg tgaggagtgg aaattgctgg
     1141 atactgcgca acaaattgtg tatcgaaatg tcatgcttga gaattacaag aacctcgtca
     1201 gtctcggata ccagttgacg aaaccggatg tgatccttag gctcgaaaag ggggaagaac
     1261 cttggctggt atctagaggc agtatcgttg ggatcctccc ccccaagaaa aagaggaagg
     1321 tatctagagg ccgcagccgc cttttggaag attttcgaaa caaccggtac cccaatttac
     1381 aactgcggga gattgctgga catataatgg aattttccca agaccagcat gggtccagat
     1441 tcattcagct gaaactggag cgtgccacac cagctgagcg ccagcttgtc ttcaatgaaa
     1501 tcctccaggc tgcctaccaa ctcatggtgg atgtgtttgg taattacgtc attcagaagt
     1561 tctttgaatt tggcagtctt gaacagaagc tggctttggc agaacggatt cgaggccacg
     1621 tcctgtcatt ggcactacag atgtatggca gccgtgttat cgagaaagct cttgagttta
     1681 ttccttcaga ccagcagaat gagatggttc gggaactaga tggccatgtc ttgaagtgtg
     1741 tgaaagatca gaatggcaat cacgtggttc agaaatgcat tgaatgtgta cagccccagt
     1801 ctttgcaatt tatcatcgat gcgtttaagg gacaggtatt tgccttatcc acacatcctt
     1861 atggctgccg agtgattcag agaatcctgg agcactgtct ccctgaccag acactcccta
     1921 ttttagagga gcttcaccag cacacagagc agctggtaca ggatcaatat ggaaattatg
     1981 taatccaaca tgtactggag cacggtcgtc ctgaggataa aagcaaaatt gtagcagaaa
     2041 tccgaggcaa tgtacttgta ttgagtcagc acaaatttgc aagcaatgtt gtggagaagt
     2101 gtgttactca cgcctcacgt acggagcgcg ctgtgctcat cgatgaggtg tgcaccatga
     2161 acgacggtcc ccacagtgcc ttatacacca tgatgaagga ccagtatgcc aactacgtgg
     2221 tccagaagat gattgacgtg gcggagccag gccagcggaa gatcgtcatg cataagatcc
     2281 ggccccacat cgcaactctt cgtaagtaca cctatggcaa gcacattctg gccaagctgg
     2341 agaagtacta catgaagaac ggtgttgact tagggtgatt aattaagaat tcgacccagc
     2401 tttcttgtac aaagtggttg atatccagca cagtggcggc cgctcgagtc tagagggccc
     2461 gcggttcgaa ggtaagccta tccctaaccc tctcctcggt ctcgattcta cgcgtaccgg
     2521 ttagcaattg ttttttcgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     2581 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2641 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     2701 aggtttttta aagcaagtaa aacctctaca aatgtggtac ttaagagggg gagaccaaag
     2761 ggcgagacgt taaggcctca cgtgacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2821 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2881 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2941 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3001 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3061 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3121 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3181 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3241 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     3301 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3361 aacaaaccac gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3421 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3481 aaactcacgt taagggattt tggtcatgcc gtctcagaag aactcgtcaa gaaggcgata
     3541 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     3601 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     3661 gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     3721 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgct
     3781 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     3841 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     3901 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     3961 catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
     4021 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     4081 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca gttcattcag
     4141 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     4201 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     4261 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcataa tattattgaa
     4321 gcatttatca gggttcgtct cgtcccggtc tcctcccatg catg
//
