LOCUS       sequence-419975-        6432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express dpdpCas13b with AAAA mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6432)
  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 6432)
  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: pzk228-pmax-SgrAI[3xFLAG]-AscI-dPspCas13b(AAAA)
FEATURES             Location/Qualifiers
     source          1..6432
                     /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"
     protein_bind    complement(4461..4485)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4538..4579
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(4605..4624)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4634..4715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4659..4678
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(4913..5501)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4993..5012)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5582..6376)
                     /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(5582..6376)
                     /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(5693..5712)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6303..6322
                     /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 cggcgcgcca tgaacatccc cgctctggtg gaaaaccaga
     1141 agaagtactt tggcacctac agcgtgatgg ccatgctgaa cgctcagacc gtgctggacc
     1201 acatccagaa ggtggccgat attgagggcg agcagaacga gaacaacgag aatctgtggt
     1261 ttcaccccgt gatgagccac ctgtacaacg ccaagaacgg ctacgacaag cagcccgaga
     1321 aaaccatgtt catcatcgag cggctgcaga gctacttccc attcctgaag atcatggccg
     1381 agaaccagag agagtacagc aacggcaagt acaagcagaa ccgcgtggaa gtgaacagca
     1441 acgacatctt cgaggtgctg aagcgcgcct tcggcgtgct gaagatgtac agggacctga
     1501 ccaacgcata caagacctac gaggaaaagc tgaacgacgg ctgcgagttc ctgaccagca
     1561 cagagcaacc tctgagcggc atgatcaaca actactacac agtggccctg cggaacatga
     1621 acgagagata cggctacaag acagaggacc tggccttcat ccaggacaag cggttcaagt
     1681 tcgtgaagga cgcctacggc aagaaaaagt cccaagtgaa taccggattc ttcctgagcc
     1741 tgcaggacta caacggcgac acacagaaga agctgcacct gagcggagtg ggaatcgccc
     1801 tgctgatctg cctgttcctg gacaagcagt acatcaacat ctttctgagc aggctgccca
     1861 tcttctccag ctacaatgcc cagagcgagg aacggcggat catcatcaga tccttcggca
     1921 tcaacagcat caagctgccc aaggaccgga tccacagcga gaagtccaac aagagcgtgg
     1981 ccatggatat gctcaacgaa gtgaagcggt gccccgacga gctgttcaca acactgtctg
     2041 ccgagaagca gtcccggttc agaatcatca gcgacgacca caatgaagtg ctgatgaagc
     2101 ggagcagcga cagattcgtg cctctgctgc tgcagtatat cgattacggc aagctgttcg
     2161 accacatcag gttccacgtg aacatgggca agctgagata cctgctggcg gccgccgcta
     2221 cctgcatcga cggccagacc agagtcagag tgatcgagca gcccctgaac ggcttcggca
     2281 gactggaaga ggccgagaca atgcggaagc aagagaacgg caccttcggc aacagcggca
     2341 tccggatcag agacttcgag aacatgaagc gggacgacgc caatcctgcc aactatccct
     2401 acatcgtgga cacctacaca cactacatcc tggaaaacaa caaggtcgag atgtttatca
     2461 acgacaaaga ggacagcgcc ccactgctgc ccgtgatcga ggatgataga tacgtggtca
     2521 agacaatccc cagctgccgg atgagcaccc tggaaattcc agccatggcc ttccacatgt
     2581 ttctgttcgg cagcaagaaa accgagaagc tgatcgtgga cgtgcacaac cggtacaaga
     2641 gactgttcca ggccatgcag aaagaagaag tgaccgccga gaatatcgcc agcttcggaa
     2701 tcgccgagag cgacctgcct cagaagatcc tggatctgat cagcggcaat gcccacggca
     2761 aggatgtgga cgccttcatc agactgaccg tggacgacat gctgaccgac accgagcgga
     2821 gaatcaagag attcaaggac gaccggaagt ccattcggag cgccgacaac aagatgggaa
     2881 agagaggctt caagcagatc tccacaggca agctggccga cttcctggcc aaggacatcg
     2941 tgctgtttca gcccagcgtg aacgatggcg agaacaagat caccggcctg aactaccgga
     3001 tcatgcagag cgccattgcc gtgtacgata gcggcgacga ttacgaggcc aagcagcagt
     3061 tcaagctgat gttcgagaag gcccggctga tcggcaaggg cacaacagag cctcatccat
     3121 ttctgtacaa ggtgttcgcc cgcagcatcc ccgccaatgc cgtcgagttc tacgagcgct
     3181 acctgatcga gcggaagttc tacctgaccg gcctgtccaa cgagatcaag aaaggcaaca
     3241 gagtggatgt gcccttcatc cggcgggacc agaacaagtg gaaaacaccc gccatgaaga
     3301 ccctgggcag aatctacagc gaggatctgc ccgtggaact gcccagacag atgttcgaca
     3361 atgagatcaa gtcccacctg aagtccctgc cacagatgga aggcatcgac ttcaacaatg
     3421 ccaacgtgac ctatctgatc gccgagtaca tgaagagagt gctggacgac gacttccaga
     3481 ccttctacca gtggaaccgc aactaccggt acatggacat gcttaagggc gagtacgaca
     3541 gaaagggctc cctgcagcac tgcttcacca gcgtggaaga gagagaaggc ctctggaaag
     3601 agcgggcctc cagaacagag cggtacagaa agcaggccag caacaagatc cgcagcaacc
     3661 ggcagatgag aaacgccagc agcgaagaga tcgagacaat cctggataag cggctgagca
     3721 acagccggaa cgagtaccag aaaagcgaga aagtgatccg gcgctacaga gtgcaggatg
     3781 ccctgctgtt tctgctggcc aaaaagaccc tgaccgaact ggccgatttc gacggcgaga
     3841 ggttcaaact gaaagaaatc atgcccgacg ccgagaaggg aatcctgagc gagatcatgc
     3901 ccatgagctt caccttcgag aaaggcggca agaagtacac catcaccagc gagggcatga
     3961 agctgaagaa ctacggcgac ttctttgtgc tggctagcga caagaggatc ggcaacctgc
     4021 tggaactcgt gggcagcgac atcgtgtcca aagaggatat catggaagag ttcaacaaat
     4081 acgaccagtg caggcccgag atcagctcca tcgtgttcaa cctggaaaag tgggccttcg
     4141 acacataccc cgagctgtct gccagagtgg accgggaaga gaaggtggac ttcaagagca
     4201 tcctgaaaat cctgctgaac aacaagaaca tcaacaaaga gcagagcgac atcctgcgga
     4261 agatccggaa cgccttcgat gcaaacaatt accccgacaa aggcgtggtg gaaatcaagg
     4321 ccctgcctga gatcgccatg agcatcaaga aggcctttgg ggagtacgcc atcatgaagg
     4381 gcggccgcgg gggaggcggt tccggtggcg gcggaagcgg aggtggagga tcagggccgg
     4441 cctgattaat taagaattcg acccagcttt cttgtacaaa gtggttgata tccagcacag
     4501 tggcggccgc tcgagtctag agggcccgcg gttcgaaggt aagcctatcc ctaaccctct
     4561 cctcggtctc gattctacgc gtaccggtta gcaattgttt tttcgatgag tttggacaaa
     4621 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     4681 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     4741 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     4801 gtggtactta agagggggag accaaagggc gagacgttaa ggcctcacgt gacatgtgag
     4861 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4921 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4981 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5041 ttccgaccct gccgcttacg ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5101 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5161 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5221 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5281 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5341 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5401 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5461 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5521 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgccgt
     5581 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     5641 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     5701 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     5761 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     5821 cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg
     5881 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     5941 tacgtgctct ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     6001 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     6061 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     6121 cagtgacaac gtcgagtaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     6181 gcgctgcctc gtcttgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     6241 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     6301 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     6361 catcttgttc aatcataata ttattgaagc atttatcagg gttcgtctcg tcccggtctc
     6421 ctcccatgca tg
//
