LOCUS       40924_30450        7685 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Middle Entry clone for Gateway containing a zebrafish 
            codon-optimized Cas9 flanked by 2 NLS and followed by inframe GFP. 
            Cas9 and GFP are separated by the sequence of a T2A self-cleaving 
            peptide..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7685)
  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 7685)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7685)
  AUTHORS   .
  TITLE     Direct Submission
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     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     99..116
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(275..302)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(394..480)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     544..560
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    576..675
                     /label=attL1
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     677..693
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        703..721
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      757..766
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             775..795
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             802..4902
                     /codon_start=1
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes MGAS15252 Type II CRISPR/Cas system"
                     /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"
     CDS             4918..4938
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4954..5007
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5014..5730
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(5770..5788)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(5817..5916)
                     /label=attL2
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(5934..5952)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5957..5973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             6086..6892
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7042..7630
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
       61 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      121 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      181 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc
      241 gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt
      301 ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg
      361 ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac
      421 cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg
      481 atgcctggca gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac
      541 gttgtaaaac gacggccagt cttaagctcg ggccccaaat aatgatttta ttttgactga
      601 tagtgacctg ttcgttgcaa caaattgatg agcaatgctt ttttataatg ccaactttgt
      661 acaaaaaagc aggcttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag
      721 ggcgaattgg gtaccgggcc ccccctcgag gtcgacgcca ccatggcttc tccacctaag
      781 aagaagagaa aggtgggaag catggataag aagtatagca tcggcctgga tattggaact
      841 aactccgtgg gttgggcagt gattacagac gactacaagg tccctagcaa gaaatttaag
      901 gtgctgggta acaccgacag gcacagcatc aagaaaaatc tgattggagc cctgctgttc
      961 ggttctggag agactgccga agcaacacgc ctgaaaagaa cagcaagaag gcgctatacc
     1021 agaaggaaga atagaatctg ttacctgcag gagattttct ctaacgaaat ggctaaggtg
     1081 gacgattcat tctttcatag gctggaggaa agtttcctgg tcgaggaaga taagaaacac
     1141 gagcgccatc ctatctttgg aaacattgtg gacgaggtcg cctatcacga aaaataccca
     1201 accatctatc atctgcgcaa gaaactggct gactctactg ataaagccga cctgagactg
     1261 atctatctgg ctctggccca catgattaag ttcaggggtc attttctgat cgagggcgat
     1321 ctgaaccccg acaattccga tgtggacaag ctgttcatcc agctggtcca gatttacaat
     1381 cagctgtttg aggaaaaccc tattaatgct tccagagtgg acgcaaaagc tatcctgtca
     1441 gccaggctgt ccaagtcacg cagactggag aacctgattg cacagctgcc cggagaaaag
     1501 aggaacggtc tgtttggaaa tctgatcgct ctgagtctgg gcctgactcc taacttcaaa
     1561 agcaattttg atctggctga ggacgccaaa ctgcagctgt caaaggacac atatgacgat
     1621 gacctggata acctgctggc acagatcgga gatcagtacg ctgacctgtt cctggctgcc
     1681 aaaaatctgt ccgacgcaat cctgctgtca gatattctga gagtgaacag cgagattaca
     1741 aaagcacctc tgagtgccag catgatcaag agatatgacg agcaccatca ggatctgacc
     1801 ctgctgaagg ctctggtcag gcagcagctg ccagagaagt acaaggaaat tttctttgat
     1861 cagtccaaga acggctacgc cggttatatc gacggaggcg catcacagga ggaattctac
     1921 aagtttatca aacctattct ggagaagatg gacggaactg aggaactgct ggtgaaactg
     1981 aatagagagg acctgctgag gaagcagcgc acatttgata acggttccat cccacaccag
     2041 attcatctgg gagagctgca cgctatcctg aggcgccagg aagacttcta cccctttctg
     2101 aaagataacc gcgagaagat cgaaaaaatt ctgaccttca gaatccctta ctatgtgggt
     2161 ccactggctc gcggaaacag cagatttgcc tggatgactc gcaaatccga ggaaaccatt
     2221 actccttgga acttcgagga agtggtcgat aagggcgcct ctgcacagtc cttcatcgag
     2281 agaatgacta attttgacaa aaacctgccc aatgagaaag tgctgcctaa gcactccctg
     2341 ctgtacgagt atttcactgt ctataacgaa ctgacaaagg tgaaatacgt caccgagggc
     2401 atgagaaagc cagccttcct gtcaggagag cagaagaaag caatcgtgga tctgctgttt
     2461 aaaaccaaca ggaaagtgac tgtcaagcag ctgaaggagg actacttcaa gaaaattgaa
     2521 tgcttcgatt ccgtggagat cagcggagtc gaagacagat ttaacgcaag cctgggcgct
     2581 taccacgatc tgctgaagat cattaaggat aaagacttcc tggacaacga ggaaaatgag
     2641 gatatcctgg aagacattgt gctgacactg accctgtttg aggacagagg aatgatcgag
     2701 gaaagactga aaacctatgc tcatctgttc gatgacaagg tgatgaaaca gctgaagaga
     2761 aggcgctaca ctggctgggg tagactgagc aggaagctga tcaacggcat tagggataaa
     2821 cagtcaggaa agacaatcct ggactttctg aaaagtgatg gcttcgccaa ccgcaatttt
     2881 atgcagctga ttcacgatga cagtctgacc ttcaaagagg acatccagaa ggctcaggtg
     2941 tctggacagg gccactccct gcatgagcag attgcaaacc tggctggaag cccagccatc
     3001 aagaaaggca ttctgcagac agtgaaaatc gtcgatgagc tggtgaaagt catgggccat
     3061 aagcccgaaa acatcgtgat tgagatggct cgcgaaaatc agacaaccca gaagggtcag
     3121 aagaacagta gagagaggat gaaaagaatc gaggaaggca ttaaggagct gggtagccag
     3181 atcctgaaag agcacccagt ggaaaacaca cagctgcaga atgagaagct gtatctgtac
     3241 tatctgcaga atggaagaga tatgtacgtg gaccaggagc tggatattaa caggctgtct
     3301 gattacgacg tggatcatat cgtcccccag agtttcatca aagatgacag cattgacaac
     3361 aaggtgctga ccaggtccga caaaaacaga ggaaaatcag ataatgtccc tagtgaggaa
     3421 gtggtcaaga aaatgaagaa ctactggaga cagctgctga atgccaaact gatcactcag
     3481 aggaagtttg ataacctgac aaaagcagag cgcggtggac tgtcagaact ggacaaagct
     3541 ggattcatca agaggcagct ggtggaaaca cgccagatca ctaaacacgt cgcacagatt
     3601 ctggatagtc gcatgaacac aaagtacgat gagaatgaca aactgatcag agaagtgaag
     3661 gtcattaccc tgaagagtaa actggtcagc gactttagga aagatttcca gttttataag
     3721 gtccgcgaga ttaacaatta tcaccatgcc catgacgcat acctgaacgc cgtggtcggt
     3781 accgcactga tcaagaaata cccaaaactg gagagcgaat tcgtgtacgg agactataag
     3841 gtgtacgatg tcagaaaaat gatcgccaag tccgagcagg aaattggaaa agctactgcc
     3901 aagtatttct tttactcaaa catcatgaat ttctttaaga cagagatcac cctggccaat
     3961 ggagaaatcc gcaaaaggcc cctgattgag acaaacggag agacaggcga aatcgtgtgg
     4021 gacaaaggca gagattttgc aaccgtgagg aaggtcctga gcatgcctca agtgaatatc
     4081 gtcaagaaaa ctgaggtgca gacaggcggt ttctcaaaag aaagtattct gccaaaacgc
     4141 aactctgata agctgatcgc tagaaagaaa gactgggacc ctaagaagta tggaggcttt
     4201 gactctccca ctgtggcata ctccgtcctg gtggtcgcta aggtggagaa gggcaaaagc
     4261 aagaaactga aatctgtcaa ggagctgctg ggtatcacaa ttatggagag aagctctttc
     4321 gagaagaacc caatcgattt tctggaggcc aaaggttata aggaagtgaa gaaagacctg
     4381 atcattaaac tgcccaagta cagtctgttt gagctggaaa acggcaggaa acgcatgctg
     4441 gcaagcgctg gagagctgca gaaaggcaat gaactggccc tgccttctaa gtacgtgaac
     4501 ttcctgtatc tggcaagcca ctacgagaag ctgaaaggat ctccagagga taacgaacag
     4561 aaacagctgt ttgtggagca gcacaagcat tatctggacg agatcattga acagattagc
     4621 gagttctcta aaagagtgat cctggccgac gcaaatctgg ataaggtcct gtctgcttac
     4681 aacaaacaca gagataagcc catcagggag caggccgaaa atatcattca tctgttcact
     4741 ctgacaaacc tgggcgcacc tgcagctttc aagtacttcg acactacaat cgatagaaag
     4801 aggtacacct ccactaagga ggtgctggac gctacactga tccatcagag tattaccggc
     4861 ctgtacgaaa caaggattga cctgtctcag ctgggtggcg actcaccggt gggatcccct
     4921 aagaagaaga gaaaggtgag gtctggtggc ggagagggca gaggcagtct gctgacatgc
     4981 ggtgacgtgg aagagaatcc cggcccttct agaatggtga gcaagggcga agagctgttc
     5041 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     5101 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     5161 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     5221 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     5281 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     5341 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     5401 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     5461 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     5521 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     5581 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     5641 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     5701 atcactctcg gcatggacga gctgtacaag taagcggccg ccaccgcggt ggagctccag
     5761 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagcaccc
     5821 agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga
     5881 acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata tcccctatag
     5941 tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct
     6001 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     6061 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     6121 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     6181 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     6241 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     6301 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     6361 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc
     6421 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     6481 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     6541 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     6601 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     6661 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     6721 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     6781 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     6841 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     6901 attggttaat tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag
     6961 ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt
     7021 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7081 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7141 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7201 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7261 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7321 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7381 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7441 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7501 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7561 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7621 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7681 tgctc
//
