LOCUS       sequence-107697-        6877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses codon optimized Cas9 in zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6877)
  AUTHORS   Yin L, Maddison LA, Li M, Kara N, LaFave MC, Varshney GK, Burgess 
            SM, Patton JG, Chen W
  TITLE     Multiplex Conditional Mutagenesis Using Transgenic Expression of 
            Cas9 and sgRNAs.
  JOURNAL   Genetics. 2015 Apr 8. pii: genetics.115.176917.
  PUBMED    25855067
REFERENCE   2  (bases 1 to 6877)
  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: "Genetics. 
            2015 Apr 8. pii: genetics.115.176917."
COMMENT     Sequence Label: pME-Cas9
FEATURES             Location/Qualifiers
     source          1..6877
                     /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     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"
     primer_bind     670..686
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        696..714
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      744..753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             762..782
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             789..4889
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes MGAS15252 Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL
                     ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             4802..4810
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             4905..4925
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4955..4975)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4955..4973)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(5002..5101)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(5119..5137)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5142..5158)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5142..5158)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5260..5279)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5271..6080
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5338..5357)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6227..6815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6716..6735
                     /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 caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggcttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
      721 tgggtaccgg gccccccctc gacgccacca tggcttctcc acctaagaag aagagaaagg
      781 tgggaagcat ggataagaag tatagcatcg gcctggatat tggaactaac tccgtgggtt
      841 gggcagtgat tacagacgac tacaaggtcc ctagcaagaa atttaaggtg ctgggtaaca
      901 ccgacaggca cagcatcaag aaaaatctga ttggagccct gctgttcggt tctggagaga
      961 ctgccgaagc aacacgcctg aaaagaacag caagaaggcg ctataccaga aggaagaata
     1021 gaatctgtta cctgcaggag attttctcta acgaaatggc taaggtggac gattcattct
     1081 ttcataggct ggaggaaagt ttcctggtcg aggaagataa gaaacacgag cgccatccta
     1141 tctttggaaa cattgtggac gaggtcgcct atcacgaaaa atacccaacc atctatcatc
     1201 tgcgcaagaa actggctgac tctactgata aagccgacct gagactgatc tatctggctc
     1261 tggcccacat gattaagttc aggggtcatt ttctgatcga gggcgatctg aaccccgaca
     1321 attccgatgt ggacaagctg ttcatccagc tggtccagat ttacaatcag ctgtttgagg
     1381 aaaaccctat taatgcttcc agagtggacg caaaagctat cctgtcagcc aggctgtcca
     1441 agtcacgcag actggagaac ctgattgcac agctgcccgg agaaaagagg aacggtctgt
     1501 ttggaaatct gatcgctctg agtctgggcc tgactcctaa cttcaaaagc aattttgatc
     1561 tggctgagga cgccaaactg cagctgtcaa aggacacata tgacgatgac ctggataacc
     1621 tgctggcaca gatcggagat cagtacgctg acctgttcct ggctgccaaa aatctgtccg
     1681 acgcaatcct gctgtcagat attctgagag tgaacagcga gattacaaaa gctcctctga
     1741 gtgccagcat gatcaagaga tatgacgagc accatcagga tctgaccctg ctgaaggctc
     1801 tggtcaggca gcagctgcca gagaagtaca aggaaatttt ctttgatcag tccaagaacg
     1861 gctacgccgg ttatatcgac ggaggcgcat cacaggagga attctacaag tttatcaaac
     1921 ctattctgga gaagatggac ggaactgagg aactgctggt gaaactgaat agagaggacc
     1981 tgctgaggaa gcagcgcaca tttgataacg gttccatccc acaccagatt catctgggag
     2041 agctgcacgc tatcctgagg cgccaggaag acttctaccc ctttctgaaa gataaccgcg
     2101 agaagatcga aaaaattctg accttcagaa tcccttacta tgtgggtcca ctggctcgcg
     2161 gaaacagcag atttgcctgg atgactcgca aatccgagga aaccattact ccttggaact
     2221 tcgaggaagt ggtcgataag ggcgcctctg cacagtcctt catcgagaga atgactaatt
     2281 ttgacaaaaa cctgcccaat gagaaagtgc tgcctaagca ctccctgctg tacgagtatt
     2341 tcactgtcta taacgaactg acaaaggtga aatacgtcac cgagggcatg agaaagccag
     2401 ccttcctgtc aggagagcag aagaaagcaa tcgtggatct gctgtttaaa accaacagga
     2461 aagtgactgt caagcagctg aaggaggact acttcaagaa aattgaatgc ttcgattccg
     2521 tggagatcag cggagtcgaa gacagattta acgcaagcct gggcgcttac cacgatctgc
     2581 tgaagatcat taaggataaa gacttcctgg acaacgagga aaatgaggat atcctggaag
     2641 acattgtgct gacactgacc ctgtttgagg acagaggaat gatcgaggaa agactgaaaa
     2701 cctatgctca tctgttcgat gacaaggtga tgaaacagct gaagagaagg cgctacactg
     2761 gctggggtag actgagcagg aagctgatca acggcattag ggataaacag tcaggaaaga
     2821 caatcctgga ctttctgaaa agtgatggct tcgccaaccg caattttatg cagctgattc
     2881 acgatgacag tctgaccttc aaagaggaca tccagaaggc tcaggtgtct ggacagggcc
     2941 actccctgca tgagcagatt gcaaacctgg ctggaagccc agccatcaag aaaggcattc
     3001 tgcagacagt gaaaatcgtc gatgagctgg tgaaagtcat gggccataag cccgaaaaca
     3061 tcgtgattga gatggctcgc gaaaatcaga caacccagaa gggtcagaag aacagtagag
     3121 agaggatgaa aagaatcgag gaaggcatta aggagctggg tagccagatc ctgaaagagc
     3181 acccagtgga aaacacacag ctgcagaatg agaagctgta tctgtactat ctgcagaatg
     3241 gaagagatat gtacgtggac caggagctgg atattaacag gctgtctgat tacgacgtgg
     3301 atcatatcgt cccccagagt ttcatcaaag atgacagcat tgacaacaag gtgctgacca
     3361 ggtccgacaa aaacagagga aaatcagata atgtccctag tgaggaagtg gtcaagaaaa
     3421 tgaagaacta ctggagacag ctgctgaatg ccaaactgat cactcagagg aagtttgata
     3481 acctgacaaa agcagagcgc ggtggactgt cagaactgga caaagctgga ttcatcaaga
     3541 ggcagctggt ggaaacacgc cagatcacta aacacgtcgc acagattctg gatagtcgca
     3601 tgaacacaaa gtacgatgag aatgacaaac tgatcagaga agtgaaggtc attaccctga
     3661 agagtaaact ggtcagcgac tttaggaaag atttccagtt ttataaggtc cgcgagatta
     3721 acaattatca ccatgcccat gacgcatacc tgaacgccgt ggtcggtacc gcactgatca
     3781 agaaataccc aaaactggag agcgaattcg tgtacggaga ctataaggtg tacgatgtca
     3841 gaaaaatgat cgccaagtcc gagcaggaaa ttggaaaagc tactgccaag tatttctttt
     3901 actcaaacat catgaatttc tttaagacag agatcaccct ggccaatgga gaaatccgca
     3961 aaaggcccct gattgagaca aacggagaga caggcgaaat cgtgtgggac aaaggcagag
     4021 attttgcaac cgtgaggaag gtcctgagca tgcctcaagt gaatatcgtc aagaaaactg
     4081 aggtgcagac aggcggtttc tcaaaagaaa gtattctgcc aaaacgcaac tctgataagc
     4141 tgatcgctag aaagaaagac tgggacccta agaagtatgg aggctttgac tctcccactg
     4201 tggcatactc cgtcctggtg gtcgctaagg tggagaaggg caaaagcaag aaactgaaat
     4261 ctgtcaagga gctgctgggt atcacaatta tggagagaag ctctttcgag aagaacccaa
     4321 tcgattttct ggaggccaaa ggttataagg aagtgaagaa agacctgatc attaaactgc
     4381 ccaagtacag tctgtttgag ctggaaaacg gcaggaaacg catgctggca agcgctggag
     4441 agctgcagaa aggcaatgaa ctggccctgc cttctaagta cgtgaacttc ctgtatctgg
     4501 caagccacta cgagaagctg aaaggatctc cagaggataa cgaacagaaa cagctgtttg
     4561 tggagcagca caagcattat ctggacgaga tcattgaaca gattagcgag ttctctaaaa
     4621 gagtgatcct ggccgacgca aatctggata aggtcctgtc tgcttacaac aaacacagag
     4681 ataagcccat cagggagcag gccgaaaata tcattcatct gttcactctg acaaacctgg
     4741 gcgcacctgc agctttcaag tacttcgaca ctacaatcga tagaaagagg tacacctcca
     4801 ctaaggaggt gctggacgct acactgatcc atcagagtat taccggcctg tacgaaacaa
     4861 ggattgacct gtctcagctg ggtggcgact caccggtgag atctcctaag aagaagagaa
     4921 aggtgtgacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     4981 tggcgtaatc atggtcatag cacccagctt tcttgtacaa agttggcatt ataagaaagc
     5041 attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt
     5101 gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc
     5161 agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa ataatatcat
     5221 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     5281 ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     5341 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga
     5401 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     5461 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     5521 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag
     5581 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     5641 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     5701 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     5761 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     5821 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     5881 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     5941 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     6001 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     6061 tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta
     6121 cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt
     6181 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6241 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6301 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6361 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6421 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6481 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6541 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6601 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6661 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6721 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6781 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6841 ggttcctggc cttttgctgg ccttttgctc acatgtt
//
