LOCUS       sequence-136517-        4859 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Clover_PUFc in pmax-DEST expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4859)
  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 4859)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pAC1447_pmax-Clover_PUFc
FEATURES             Location/Qualifiers
     source          1..4859
                     /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             join(1008..1010,1011..1013,1014..1724)
                     /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(1053..1074)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1661..1682
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1740..1758)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1741..1758)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1797..1817
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2889..2913)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2966..3007
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3033..3052)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3062..3143
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3087..3106
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3341..3928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3421..3440)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4009..4803)
                     /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(4009..4803)
                     /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(4120..4139)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4730..4749
                     /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 gtgagcaagg
     1021 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1081 gccacaagtt cagcgtccgc ggcgagggcg agggcgatgc caccaacggc aagctgaccc
     1141 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct
     1201 tcggctacgg cgtggcctgc ttcagccgct accccgacca catgaagcag cacgacttct
     1261 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctctttc aaggacgacg
     1321 gtacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1381 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1441 acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc atcaaggcta
     1501 acttcaagat ccgccacaac gttgaggacg gcagcgtgca gctcgccgac cactaccagc
     1561 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagccatc
     1621 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1681 tgaccgccgc cgggattaca catggcatgg acgagctgta caagtctaga gggccctatt
     1741 ctatagtgtc acctaaatgc ggaggcggcg gtagcgggcc ggccgggatc ctccccccca
     1801 agaaaaagag gaaggtatct agaggccgca gccgcctttt ggaagatttt cgaaacaacc
     1861 ggtaccccaa tttacaactg cgggagattg ctggacatat aatggaattt tcccaagacc
     1921 agcatgggtc cagattcatt cagctgaaac tggagcgtgc cacaccagct gagcgccagc
     1981 ttgtcttcaa tgaaatcctc caggctgcct accaactcat ggtggatgtg tttggtaatt
     2041 acgtcattca gaagttcttt gaatttggca gtcttgaaca gaagctggct ttggcagaac
     2101 ggattcgagg ccacgtcctg tcattggcac tacagatgta tggcagccgt gttatcgaga
     2161 aagctcttga gtttattcct tcagaccagc agaatgagat ggttcgggaa ctagatggcc
     2221 atgtcttgaa gtgtgtgaaa gatcagaatg gcaatcacgt ggttcagaaa tgcattgaat
     2281 gtgtacagcc ccagtctttg caatttatca tcgacgcgtt taagggacag gtatttgcct
     2341 tatccacaca tccttatggc tgccgagtga ttcagagaat cctggagcac tgtctccctg
     2401 accagacact ccctatttta gaggagcttc accagcacac agagcagctg gtacaggatc
     2461 aatatggaag ttatgtaatc gaacatgtac tggagcacgg tcgtcctgag gataaaagca
     2521 aaattgtagc agaaatccga ggcaatgtac ttgtattgag tcagcacaaa tttgcaaaca
     2581 atgttgtgca gaagtgtgtt actcacgcct cacgtacgga gcgcgctgtg ctcatcgacg
     2641 aggtgtgcac catgaacgac ggtccccaca gtgccttata caccatgatg aaggaccagt
     2701 atgccaacta cgtggtccag aagatgattg acgtggcgga gccaggccag cggaagatcg
     2761 tcatgcataa gatccggccc cacatcgcaa ctcttcgtaa gtacacctat ggcaagcaca
     2821 ttctggccaa gctggagaag tactacatga agaacggtgt tgacttaggg tgattaatta
     2881 agaattcgac ccagctttct tgtacaaagt ggttgatatc cagcacagtg gcggccgctc
     2941 gagtctagag ggcccgcggt tcgaaggtaa gcctatccct aaccctctcc tcggtctcga
     3001 ttctacgcgt accggttagc aattgttttt tcgatgagtt tggacaaacc acaactagaa
     3061 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     3121 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     3181 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtacttaag
     3241 agggggagac caaagggcga gacgttaagg cctcacgtga catgtgagca aaaggccagc
     3301 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3361 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3421 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3481 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3541 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3601 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3661 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3721 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3781 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3841 gctcttgatc cggcaaacaa accacgctgg tagcggtggt ttttttgttt gcaagcagca
     3901 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3961 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgccgtctc agaagaactc
     4021 gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac
     4081 gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc
     4141 tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg
     4201 gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc
     4261 gccgtcgggc atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg
     4321 ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc
     4381 gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg
     4441 ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag
     4501 atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc
     4561 gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc
     4621 ttgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg
     4681 cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata
     4741 gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat
     4801 cataatatta ttgaagcatt tatcagggtt cgtctcgtcc cggtctcctc ccatgcatg
//
