LOCUS       sequence-420041-        7392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express dpdpCas13b with AAAA mutation and Clover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7392)
  AUTHORS   Liu Z, Jillette N, Robson P, Cheng AW
  TITLE     Simultaneous multifunctional transcriptome engineering by CRISPR RNA
            scaffold.
  JOURNAL   Nucleic Acids Res. 2023 Aug 11;51(14):e77. doi: 10.1093/nar/gkad547.
  PUBMED    37395412
REFERENCE   2  (bases 1 to 7392)
  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"; journalName: "Nucleic 
            Acids Res."; date: "2023-08-11"; volume: "51(14):e77. doi"; pages: "
            10.1093/nar/gkad547"
COMMENT     Sequence Label: pmax-3xFLAG_3xNLS_dPspCas13b(AAAA)_2xNLS-Clover
FEATURES             Location/Qualifiers
     source          1..7392
                     /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             1011..1076
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1101..1121
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1125..1145
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1149..1169
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4545..4565
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4569..4589
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4644..4646,4647..4649,4650..5360)
                     /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(4689..4710)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     5297..5318
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(5376..5394)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5377..5394)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    complement(5421..5445)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5498..5539
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(5565..5584)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    5594..5675
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5619..5638
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(5873..6461)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5953..5972)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6542..7336)
                     /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(6542..7336)
                     /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(6653..6672)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7263..7282
                     /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 gactacaagg
     1021 atcacgacgg tgactataag gatcatgaca tcgactataa ggacgatgac gataagatcg
     1081 atggcggagg cggatctgat ccaaaaaaga agagaaaggt agatccaaaa aagaagagaa
     1141 aggtagatcc aaaaaagaag agaaaggtag gatctaccgg atctagaaac gatggtggtg
     1201 gtggaagcgg gggtgggggc agcggtggag ggggaagcgg gcgcgccatg aacatccccg
     1261 ctctggtgga aaaccagaag aagtactttg gcacctacag cgtgatggcc atgctgaacg
     1321 ctcagaccgt gctggaccac atccagaagg tggccgatat tgagggcgag cagaacgaga
     1381 acaacgagaa tctgtggttt caccccgtga tgagccacct gtacaacgcc aagaacggct
     1441 acgacaagca gcccgagaaa accatgttca tcatcgagcg gctgcagagc tacttcccat
     1501 tcctgaagat catggccgag aaccagagag agtacagcaa cggcaagtac aagcagaacc
     1561 gcgtggaagt gaacagcaac gacatcttcg aggtgctgaa gcgcgccttc ggcgtgctga
     1621 agatgtacag ggacctgacc aacgcataca agacctacga ggaaaagctg aacgacggct
     1681 gcgagttcct gaccagcaca gagcaacctc tgagcggcat gatcaacaac tactacacag
     1741 tggccctgcg gaacatgaac gagagatacg gctacaagac agaggacctg gccttcatcc
     1801 aggacaagcg gttcaagttc gtgaaggacg cctacggcaa gaaaaagtcc caagtgaata
     1861 ccggattctt cctgagcctg caggactaca acggcgacac acagaagaag ctgcacctga
     1921 gcggagtggg aatcgccctg ctgatctgcc tgttcctgga caagcagtac atcaacatct
     1981 ttctgagcag gctgcccatc ttctccagct acaatgccca gagcgaggaa cggcggatca
     2041 tcatcagatc cttcggcatc aacagcatca agctgcccaa ggaccggatc cacagcgaga
     2101 agtccaacaa gagcgtggcc atggatatgc tcaacgaagt gaagcggtgc cccgacgagc
     2161 tgttcacaac actgtctgcc gagaagcagt cccggttcag aatcatcagc gacgaccaca
     2221 atgaagtgct gatgaagcgg agcagcgaca gattcgtgcc tctgctgctg cagtatatcg
     2281 attacggcaa gctgttcgac cacatcaggt tccacgtgaa catgggcaag ctgagatacc
     2341 tgctggcggc cgccgctacc tgcatcgacg gccagaccag agtcagagtg atcgagcagc
     2401 ccctgaacgg cttcggcaga ctggaagagg ccgagacaat gcggaagcaa gagaacggca
     2461 ccttcggcaa cagcggcatc cggatcagag acttcgagaa catgaagcgg gacgacgcca
     2521 atcctgccaa ctatccctac atcgtggaca cctacacaca ctacatcctg gaaaacaaca
     2581 aggtcgagat gtttatcaac gacaaagagg acagcgcccc actgctgccc gtgatcgagg
     2641 atgatagata cgtggtcaag acaatcccca gctgccggat gagcaccctg gaaattccag
     2701 ccatggcctt ccacatgttt ctgttcggca gcaagaaaac cgagaagctg atcgtggacg
     2761 tgcacaaccg gtacaagaga ctgttccagg ccatgcagaa agaagaagtg accgccgaga
     2821 atatcgccag cttcggaatc gccgagagcg acctgcctca gaagatcctg gatctgatca
     2881 gcggcaatgc ccacggcaag gatgtggacg ccttcatcag actgaccgtg gacgacatgc
     2941 tgaccgacac cgagcggaga atcaagagat tcaaggacga ccggaagtcc attcggagcg
     3001 ccgacaacaa gatgggaaag agaggcttca agcagatctc cacaggcaag ctggccgact
     3061 tcctggccaa ggacatcgtg ctgtttcagc ccagcgtgaa cgatggcgag aacaagatca
     3121 ccggcctgaa ctaccggatc atgcagagcg ccattgccgt gtacgatagc ggcgacgatt
     3181 acgaggccaa gcagcagttc aagctgatgt tcgagaaggc ccggctgatc ggcaagggca
     3241 caacagagcc tcatccattt ctgtacaagg tgttcgcccg cagcatcccc gccaatgccg
     3301 tcgagttcta cgagcgctac ctgatcgagc ggaagttcta cctgaccggc ctgtccaacg
     3361 agatcaagaa aggcaacaga gtggatgtgc ccttcatccg gcgggaccag aacaagtgga
     3421 aaacacccgc catgaagacc ctgggcagaa tctacagcga ggatctgccc gtggaactgc
     3481 ccagacagat gttcgacaat gagatcaagt cccacctgaa gtccctgcca cagatggaag
     3541 gcatcgactt caacaatgcc aacgtgacct atctgatcgc cgagtacatg aagagagtgc
     3601 tggacgacga cttccagacc ttctaccagt ggaaccgcaa ctaccggtac atggacatgc
     3661 ttaagggcga gtacgacaga aagggctccc tgcagcactg cttcaccagc gtggaagaga
     3721 gagaaggcct ctggaaagag cgggcctcca gaacagagcg gtacagaaag caggccagca
     3781 acaagatccg cagcaaccgg cagatgagaa acgccagcag cgaagagatc gagacaatcc
     3841 tggataagcg gctgagcaac agccggaacg agtaccagaa aagcgagaaa gtgatccggc
     3901 gctacagagt gcaggatgcc ctgctgtttc tgctggccaa aaagaccctg accgaactgg
     3961 ccgatttcga cggcgagagg ttcaaactga aagaaatcat gcccgacgcc gagaagggaa
     4021 tcctgagcga gatcatgccc atgagcttca ccttcgagaa aggcggcaag aagtacacca
     4081 tcaccagcga gggcatgaag ctgaagaact acggcgactt ctttgtgctg gctagcgaca
     4141 agaggatcgg caacctgctg gaactcgtgg gcagcgacat cgtgtccaaa gaggatatca
     4201 tggaagagtt caacaaatac gaccagtgca ggcccgagat cagctccatc gtgttcaacc
     4261 tggaaaagtg ggccttcgac acataccccg agctgtctgc cagagtggac cgggaagaga
     4321 aggtggactt caagagcatc ctgaaaatcc tgctgaacaa caagaacatc aacaaagagc
     4381 agagcgacat cctgcggaag atccggaacg ccttcgatgc aaacaattac cccgacaaag
     4441 gcgtggtgga aatcaaggcc ctgcctgaga tcgccatgag catcaagaag gcctttgggg
     4501 agtacgccat catgaagggc ggccgcggag ggggtggaag tgacccaaag aagaagcgca
     4561 aagtggatcc taaaaagaaa agaaaggtag ggggaggcgg ttccggtggc ggcggaagcg
     4621 gaggtggagg atcagggccg gccatggtga gcaagggcga ggagctgttc accggggtgg
     4681 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtccgcggcg
     4741 agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca
     4801 agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcgtg gcctgcttca
     4861 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     4921 acgtccagga gcgcaccatc tctttcaagg acgacggtac ctacaagacc cgcgccgagg
     4981 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     5041 aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac aacgtctata
     5101 tcacggccga caagcagaag aacggcatca aggctaactt caagatccgc cacaacgttg
     5161 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     5221 ccgtgctgct gcccgacaac cactacctga gccatcagtc cgccctgagc aaagacccca
     5281 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg attacacatg
     5341 gcatggacga gctgtacaag tctagagggc cctattctat agtgtcacct aaatgcatcg
     5401 attgattaat taagaattcg acccagcttt cttgtacaaa gtggttgata tccagcacag
     5461 tggcggccgc tcgagtctag agggcccgcg gttcgaaggt aagcctatcc ctaaccctct
     5521 cctcggtctc gattctacgc gtaccggtta gcaattgttt tttcgatgag tttggacaaa
     5581 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     5641 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     5701 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     5761 gtggtactta agagggggag accaaagggc gagacgttaa ggcctcacgt gacatgtgag
     5821 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5881 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5941 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6001 ttccgaccct gccgcttacg ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6061 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6121 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6181 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6241 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6301 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6361 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6421 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6481 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgccgt
     6541 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     6601 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     6661 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     6721 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     6781 cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg
     6841 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     6901 tacgtgctct ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     6961 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     7021 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     7081 cagtgacaac gtcgagtaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     7141 gcgctgcctc gtcttgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     7201 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     7261 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     7321 catcttgttc aatcataata ttattgaagc atttatcagg gttcgtctcg tcccggtctc
     7381 ctcccatgca tg
//
