LOCUS       sequence-176267-        6907 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle Entry clone for Gateway containing a zebrafish 
            codon-optimized Cas9 flanked by 2 NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6907)
  AUTHORS   Ablain J, Durand EM, Yang S, Zhou Y, Zon LI
  TITLE     A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in 
            Zebrafish.
  JOURNAL   Dev Cell. 2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 
            10.1016/j.devcel.2015.01.032.
  PUBMED    25752963
REFERENCE   2  (bases 1 to 6907)
  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: "Dev Cell. 
            2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 
            10.1016/j.devcel.2015.01.032."
COMMENT     Sequence Label: pME-Cas9
FEATURES             Location/Qualifiers
     source          1..6907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..21
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             28..4128
                     /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             4041..4049
                     /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             4144..4164
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4218..4238)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4218..4236)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(4265..4364)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(4382..4400)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4405..4421)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4405..4421)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4523..4542)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4534..5343
                     /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(4601..4620)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5490..6078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5979..5998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6232..6249
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6408..6435)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6527..6613)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     6662..6684
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6677..6693
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6709..6808
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     6810..6826
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6836..6854
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6890..6899
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 cctaagaaga agagaaaggt gggaagcatg gataagaagt atagcatcgg cctggatatt
       61 ggaactaact ccgtgggttg ggcagtgatt acagacgact acaaggtccc tagcaagaaa
      121 tttaaggtgc tgggtaacac cgacaggcac agcatcaaga aaaatctgat tggagccctg
      181 ctgttcggtt ctggagagac tgccgaagca acacgcctga aaagaacagc aagaaggcgc
      241 tataccagaa ggaagaatag aatctgttac ctgcaggaga ttttctctaa cgaaatggct
      301 aaggtggacg attcattctt tcataggctg gaggaaagtt tcctggtcga ggaagataag
      361 aaacacgagc gccatcctat ctttggaaac attgtggacg aggtcgccta tcacgaaaaa
      421 tacccaacca tctatcatct gcgcaagaaa ctggctgact ctactgataa agccgacctg
      481 agactgatct atctggctct ggcccacatg attaagttca ggggtcattt tctgatcgag
      541 ggcgatctga accccgacaa ttccgatgtg gacaagctgt tcatccagct ggtccagatt
      601 tacaatcagc tgtttgagga aaaccctatt aatgcttcca gagtggacgc aaaagctatc
      661 ctgtcagcca ggctgtccaa gtcacgcaga ctggagaacc tgattgcaca gctgcccgga
      721 gaaaagagga acggtctgtt tggaaatctg atcgctctga gtctgggcct gactcctaac
      781 ttcaaaagca attttgatct ggctgaggac gccaaactgc agctgtcaaa ggacacatat
      841 gacgatgacc tggataacct gctggcacag atcggagatc agtacgctga cctgttcctg
      901 gctgccaaaa atctgtccga cgcaatcctg ctgtcagata ttctgagagt gaacagcgag
      961 attacaaaag cacctctgag tgccagcatg atcaagagat atgacgagca ccatcaggat
     1021 ctgaccctgc tgaaggctct ggtcaggcag cagctgccag agaagtacaa ggaaattttc
     1081 tttgatcagt ccaagaacgg ctacgccggt tatatcgacg gaggcgcatc acaggaggaa
     1141 ttctacaagt ttatcaaacc tattctggag aagatggacg gaactgagga actgctggtg
     1201 aaactgaata gagaggacct gctgaggaag cagcgcacat ttgataacgg ttccatccca
     1261 caccagattc atctgggaga gctgcacgct atcctgaggc gccaggaaga cttctacccc
     1321 tttctgaaag ataaccgcga gaagatcgaa aaaattctga ccttcagaat cccttactat
     1381 gtgggtccac tggctcgcgg aaacagcaga tttgcctgga tgactcgcaa atccgaggaa
     1441 accattactc cttggaactt cgaggaagtg gtcgataagg gcgcctctgc acagtccttc
     1501 atcgagagaa tgactaattt tgacaaaaac ctgcccaatg agaaagtgct gcctaagcac
     1561 tccctgctgt acgagtattt cactgtctat aacgaactga caaaggtgaa atacgtcacc
     1621 gagggcatga gaaagccagc cttcctgtca ggagagcaga agaaagcaat cgtggatctg
     1681 ctgtttaaaa ccaacaggaa agtgactgtc aagcagctga aggaggacta cttcaagaaa
     1741 attgaatgct tcgattccgt ggagatcagc ggagtcgaag acagatttaa cgcaagcctg
     1801 ggcgcttacc acgatctgct gaagatcatt aaggataaag acttcctgga caacgaggaa
     1861 aatgaggata tcctggaaga cattgtgctg acactgaccc tgtttgagga cagaggaatg
     1921 atcgaggaaa gactgaaaac ctatgctcat ctgttcgatg acaaggtgat gaaacagctg
     1981 aagagaaggc gctacactgg ctggggtaga ctgagcagga agctgatcaa cggcattagg
     2041 gataaacagt caggaaagac aatcctggac tttctgaaaa gtgatggctt cgccaaccgc
     2101 aattttatgc agctgattca cgatgacagt ctgaccttca aagaggacat ccagaaggct
     2161 caggtgtctg gacagggcca ctccctgcat gagcagattg caaacctggc tggaagccca
     2221 gccatcaaga aaggcattct gcagacagtg aaaatcgtcg atgagctggt gaaagtcatg
     2281 ggccataagc ccgaaaacat cgtgattgag atggctcgcg aaaatcagac aacccagaag
     2341 ggtcagaaga acagtagaga gaggatgaaa agaatcgagg aaggcattaa ggagctgggt
     2401 agccagatcc tgaaagagca cccagtggaa aacacacagc tgcagaatga gaagctgtat
     2461 ctgtactatc tgcagaatgg aagagatatg tacgtggacc aggagctgga tattaacagg
     2521 ctgtctgatt acgacgtgga tcatatcgtc ccccagagtt tcatcaaaga tgacagcatt
     2581 gacaacaagg tgctgaccag gtccgacaaa aacagaggaa aatcagataa tgtccctagt
     2641 gaggaagtgg tcaagaaaat gaagaactac tggagacagc tgctgaatgc caaactgatc
     2701 actcagagga agtttgataa cctgacaaaa gcagagcgcg gtggactgtc agaactggac
     2761 aaagctggat tcatcaagag gcagctggtg gaaacacgcc agatcactaa acacgtcgca
     2821 cagattctgg atagtcgcat gaacacaaag tacgatgaga atgacaaact gatcagagaa
     2881 gtgaaggtca ttaccctgaa gagtaaactg gtcagcgact ttaggaaaga tttccagttt
     2941 tataaggtcc gcgagattaa caattatcac catgcccatg acgcatacct gaacgccgtg
     3001 gtcggtaccg cactgatcaa gaaataccca aaactggaga gcgaattcgt gtacggagac
     3061 tataaggtgt acgatgtcag aaaaatgatc gccaagtccg agcaggaaat tggaaaagct
     3121 actgccaagt atttctttta ctcaaacatc atgaatttct ttaagacaga gatcaccctg
     3181 gccaatggag aaatccgcaa aaggcccctg attgagacaa acggagagac aggcgaaatc
     3241 gtgtgggaca aaggcagaga ttttgcaacc gtgaggaagg tcctgagcat gcctcaagtg
     3301 aatatcgtca agaaaactga ggtgcagaca ggcggtttct caaaagaaag tattctgcca
     3361 aaacgcaact ctgataagct gatcgctaga aagaaagact gggaccctaa gaagtatgga
     3421 ggctttgact ctcccactgt ggcatactcc gtcctggtgg tcgctaaggt ggagaagggc
     3481 aaaagcaaga aactgaaatc tgtcaaggag ctgctgggta tcacaattat ggagagaagc
     3541 tctttcgaga agaacccaat cgattttctg gaggccaaag gttataagga agtgaagaaa
     3601 gacctgatca ttaaactgcc caagtacagt ctgtttgagc tggaaaacgg caggaaacgc
     3661 atgctggcaa gcgctggaga gctgcagaaa ggcaatgaac tggccctgcc ttctaagtac
     3721 gtgaacttcc tgtatctggc aagccactac gagaagctga aaggatctcc agaggataac
     3781 gaacagaaac agctgtttgt ggagcagcac aagcattatc tggacgagat cattgaacag
     3841 attagcgagt tctctaaaag agtgatcctg gccgacgcaa atctggataa ggtcctgtct
     3901 gcttacaaca aacacagaga taagcccatc agggagcagg ccgaaaatat cattcatctg
     3961 ttcactctga caaacctggg cgcacctgca gctttcaagt acttcgacac tacaatcgat
     4021 agaaagaggt acacctccac taaggaggtg ctggacgcta cactgatcca tcagagtatt
     4081 accggcctgt acgaaacaag gattgacctg tctcagctgg gtggcgactc accggtgaga
     4141 tctcctaaga agaagagaaa ggtgtgaccg cggaatctag agcggccgcc accgcggtgg
     4201 agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     4261 tagcacccag ctttcttgta caaagttggc attataagaa agcattgctt atcaatttgt
     4321 tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgatatc
     4381 ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct
     4441 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg
     4501 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg
     4561 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     4621 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag
     4681 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     4741 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     4801 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa
     4861 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     4921 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     4981 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     5041 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     5101 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     5161 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     5221 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     5281 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     5341 taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac
     5401 ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca
     5461 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5521 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5581 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5641 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5701 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5761 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5821 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5881 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5941 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6001 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6061 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6121 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6181 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     6241 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     6301 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     6361 gcaattaata cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg
     6421 tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc
     6481 accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca
     6541 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct
     6601 ttcgttttat ttgatgcctg gcagttccct actctcgcgt taacgctagc atggatgttt
     6661 tcccagtcac gacgttgtaa aacgacggcc agtcttaagc tcgggcccca aataatgatt
     6721 ttattttgac tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata
     6781 atgccaactt tgtacaaaaa agcaggcttg taaaacgacg gccagtgagc gcgcgtaata
     6841 cgactcacta tagggcgaat tgggtaccgg gccccccctc gaggtcgacg ccaccatggc
     6901 ttctcca
//
