pME-Cas9-T2A-GFP

63155 Danio rerio Bacterial Expression

Plasmid Information

Purpose
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.
Depositor
Leonard Zon
Article
Ablain et al Dev Cell. 2015 Mar 4. pii: S1534-5807(15
Insert
Cas9-T2A-GFP
Available Since
April 1, 2015
Availability
Available

Sequence Information

Length
7,685 bp
Topology
circular
Molecule Type
other DNA
View on AddGene.org

Features

CDS 7
Label Gene Product Position Strand
SV40 NLS — nuclear localization signal of SV40 (si… 774..795 Fwd
DNA Sequence
CCTAAGAAGAAGAGAAAGGTG
Protein Sequence
PKKKRKV
Cas9 — Cas9 (Csn1) endonuclease from the Strep… 801..4902 Fwd
DNA Sequence
ATGGATAAGAAGTATAGCATCGGCCTGGATATTGGAACTAACTCCGTGGGTTGGGCAGTGATTACAGACGACTACAAGGTCCCTAGCAAGAAATTTAAGGTGCTGGGTAACACCGACAGGCACAGCATCAAGAAAAATCTGATTGGAGCCCTGCTGTTCGGTTCTGGAGAGACTGCCGAAGCAACACGCCTGAAAAGAACAGCAAGAAGGCGCTATACCAGAAGGAAGAATAGAATCTGTTACCTGCAGGAGATTTTCTCTAACGAAATGGCTAAGGTGGACGATTCATTCTTTCATAGGCTGGAGGAAAGTTTCCTGGTCGAGGAAGATAAGAAACACGAGCGCCATCCTATCTTTGGAAACATTGTGGACGAGGTCGCCTATCACGAAAAATACCCAACCATCTATCATCTGCGCAAGAAACTGGCTGACTCTACTGATAAAGCCGACCTGAGACTGATCTATCTGGCTCTGGCCCACATGATTAAGTTCAGGGGTCATTTTCTGATCGAGGGCGATCTGAACCCCGACAATTCCGATGTGGACAAGCTGTTCATCCAGCTGGTCCAGATTTACAATCAGCTGTTTGAGGAAAACCCTATTAATGCTTCCAGAGTGGACGCAAAAGCTATCCTGTCAGCCAGGCTGTCCAAGTCACGCAGACTGGAGAACCTGATTGCACAGCTGCCCGGAGAAAAGAGGAACGGTCTGTTTGGAAATCTGATCGCTCTGAGTCTGGGCCTGACTCCTAACTTCAAAAGCAATTTTGATCTGGCTGAGGACGCCAAACTGCAGCTGTCAAAGGACACATATGACGATGACCTGGATAACCTGCTGGCACAGATCGGAGATCAGTACGCTGACCTGTTCCTGGCTGCCAAAAATCTGTCCGACGCAATCCTGCTGTCAGATATTCTGAGAGTGAACAGCGAGATTACAAAAGCACCTCTGAGTGCCAGCATGATCAAGAGATATGACGAGCACCATCAGGATCTGACCCTGCTGAAGGCTCTGGTCAGGCAGCAGCTGCCAGAGAAGTACAAGGAAATTTTCTTTGATCAGTCCAAGAACGGCTACGCCGGTTATATCGACGGAGGCGCATCACAGGAGGAATTCTACAAGTTTATCAAACCTATTCTGGAGAAGATGGACGGAACTGAGGAACTGCTGGTGAAACTGAATAGAGAGGACCTGCTGAGGAAGCAGCGCACATTTGATAACGGTTCCATCCCACACCAGATTCATCTGGGAGAGCTGCACGCTATCCTGAGGCGCCAGGAAGACTTCTACCCCTTTCTGAAAGATAACCGCGAGAAGATCGAAAAAATTCTGACCTTCAGAATCCCTTACTATGTGGGTCCACTGGCTCGCGGAAACAGCAGATTTGCCTGGATGACTCGCAAATCCGAGGAAACCATTACTCCTTGGAACTTCGAGGAAGTGGTCGATAAGGGCGCCTCTGCACAGTCCTTCATCGAGAGAATGACTAATTTTGACAAAAACCTGCCCAATGAGAAAGTGCTGCCTAAGCACTCCCTGCTGTACGAGTATTTCACTGTCTATAACGAACTGACAAAGGTGAAATACGTCACCGAGGGCATGAGAAAGCCAGCCTTCCTGTCAGGAGAGCAGAAGAAAGCAATCGTGGATCTGCTGTTTAAAACCAACAGGAAAGTGACTGTCAAGCAGCTGAAGGAGGACTACTTCAAGAAAATTGAATGCTTCGATTCCGTGGAGATCAGCGGAGTCGAAGACAGATTTAACGCAAGCCTGGGCGCTTACCACGATCTGCTGAAGATCATTAAGGATAAAGACTTCCTGGACAACGAGGAAAATGAGGATATCCTGGAAGACATTGTGCTGACACTGACCCTGTTTGAGGACAGAGGAATGATCGAGGAAAGACTGAAAACCTATGCTCATCTGTTCGATGACAAGGTGATGAAACAGCTGAAGAGAAGGCGCTACACTGGCTGGGGTAGACTGAGCAGGAAGCTGATCAACGGCATTAGGGATAAACAGTCAGGAAAGACAATCCTGGACTTTCTGAAAAGTGATGGCTTCGCCAACCGCAATTTTATGCAGCTGATTCACGATGACAGTCTGACCTTCAAAGAGGACATCCAGAAGGCTCAGGTGTCTGGACAGGGCCACTCCCTGCATGAGCAGATTGCAAACCTGGCTGGAAGCCCAGCCATCAAGAAAGGCATTCTGCAGACAGTGAAAATCGTCGATGAGCTGGTGAAAGTCATGGGCCATAAGCCCGAAAACATCGTGATTGAGATGGCTCGCGAAAATCAGACAACCCAGAAGGGTCAGAAGAACAGTAGAGAGAGGATGAAAAGAATCGAGGAAGGCATTAAGGAGCTGGGTAGCCAGATCCTGAAAGAGCACCCAGTGGAAAACACACAGCTGCAGAATGAGAAGCTGTATCTGTACTATCTGCAGAATGGAAGAGATATGTACGTGGACCAGGAGCTGGATATTAACAGGCTGTCTGATTACGACGTGGATCATATCGTCCCCCAGAGTTTCATCAAAGATGACAGCATTGACAACAAGGTGCTGACCAGGTCCGACAAAAACAGAGGAAAATCAGATAATGTCCCTAGTGAGGAAGTGGTCAAGAAAATGAAGAACTACTGGAGACAGCTGCTGAATGCCAAACTGATCACTCAGAGGAAGTTTGATAACCTGACAAAAGCAGAGCGCGGTGGACTGTCAGAACTGGACAAAGCTGGATTCATCAAGAGGCAGCTGGTGGAAACACGCCAGATCACTAAACACGTCGCACAGATTCTGGATAGTCGCATGAACACAAAGTACGATGAGAATGACAAACTGATCAGAGAAGTGAAGGTCATTACCCTGAAGAGTAAACTGGTCAGCGACTTTAGGAAAGATTTCCAGTTTTATAAGGTCCGCGAGATTAACAATTATCACCATGCCCATGACGCATACCTGAACGCCGTGGTCGGTACCGCACTGATCAAGAAATACCCAAAACTGGAGAGCGAATTCGTGTACGGAGACTATAAGGTGTACGATGTCAGAAAAATGATCGCCAAGTCCGAGCAGGAAATTGGAAAAGCTACTGCCAAGTATTTCTTTTACTCAAACATCATGAATTTCTTTAAGACAGAGATCACCCTGGCCAATGGAGAAATCCGCAAAAGGCCCCTGATTGAGACAAACGGAGAGACAGGCGAAATCGTGTGGGACAAAGGCAGAGATTTTGCAACCGTGAGGAAGGTCCTGAGCATGCCTCAAGTGAATATCGTCAAGAAAACTGAGGTGCAGACAGGCGGTTTCTCAAAAGAAAGTATTCTGCCAAAACGCAACTCTGATAAGCTGATCGCTAGAAAGAAAGACTGGGACCCTAAGAAGTATGGAGGCTTTGACTCTCCCACTGTGGCATACTCCGTCCTGGTGGTCGCTAAGGTGGAGAAGGGCAAAAGCAAGAAACTGAAATCTGTCAAGGAGCTGCTGGGTATCACAATTATGGAGAGAAGCTCTTTCGAGAAGAACCCAATCGATTTTCTGGAGGCCAAAGGTTATAAGGAAGTGAAGAAAGACCTGATCATTAAACTGCCCAAGTACAGTCTGTTTGAGCTGGAAAACGGCAGGAAACGCATGCTGGCAAGCGCTGGAGAGCTGCAGAAAGGCAATGAACTGGCCCTGCCTTCTAAGTACGTGAACTTCCTGTATCTGGCAAGCCACTACGAGAAGCTGAAAGGATCTCCAGAGGATAACGAACAGAAACAGCTGTTTGTGGAGCAGCACAAGCATTATCTGGACGAGATCATTGAACAGATTAGCGAGTTCTCTAAAAGAGTGATCCTGGCCGACGCAAATCTGGATAAGGTCCTGTCTGCTTACAACAAACACAGAGATAAGCCCATCAGGGAGCAGGCCGAAAATATCATTCATCTGTTCACTCTGACAAACCTGGGCGCACCTGCAGCTTTCAAGTACTTCGACACTACAATCGATAGAAAGAGGTACACCTCCACTAAGGAGGTGCTGGACGCTACACTGATCCATCAGAGTATTACCGGCCTGTACGAAACAAGGATTGACCTGTCTCAGCTGGGTGGCGAC
Protein Sequence
MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
SV40 NLS — nuclear localization signal of SV40 (si… 4917..4938 Fwd
DNA Sequence
CCTAAGAAGAAGAGAAAGGTG
Protein Sequence
PKKKRKV
T2A — 2A peptide from Thosea asigna virus cap… 4953..5007 Fwd
DNA Sequence
GAGGGCAGAGGCAGTCTGCTGACATGCGGTGACGTGGAAGAGAATCCCGGCCCT
Protein Sequence
EGRGSLLTCGDVEENPGP
EGFP — the original enhanced GFP (Yang et al.,… 5013..5733 Fwd
DNA Sequence
ATGGTGAGCAAGGGCGAAGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
Protein Sequence
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
EGFP — the original enhanced GFP (Yang et al.,… 5013..5730 Fwd
DNA Sequence
ATGGTGAGCAAGGGCGAAGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG
Protein Sequence
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
KanR aph(3')-Ia aminoglycoside phosphotransferase 6085..6895 Fwd
DNA Sequence
ATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA
Protein Sequence
MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF
RBS 1
Label Gene Product Position Strand
Shine-Dalgarno sequence — — 4814..4823 Fwd
DNA Sequence
TAAGGAGGT
promoter 3
Label Gene Product Position Strand
T7 promoter — — 702..721 Fwd
DNA Sequence
TAATACGACTCACTATAGG
T3 promoter — — 5769..5788 Rev
DNA Sequence
AATTAACCCTCACTAAAGG
T7 promoter — — 5933..5952 Rev
DNA Sequence
TAATACGACTCACTATAGG
protein_bind 2
Label Gene Product Position Strand
attL1 mutant version of attL — 575..675 Fwd
DNA Sequence
CAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCT
attL2 mutant version of attL — 5816..5916 Rev
DNA Sequence
CAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATAAGCAATGCTTTCTTATAATGCCAACTTTGTACAAGAAAGCTGGGT
rep_origin 1
Label Gene Product Position Strand
ori — — 7041..7630 Fwd
DNA Sequence
TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
terminator 2
Label Gene Product Position Strand
rrnB T2 terminator — — 274..302 Rev
DNA Sequence
AGAAGGCCATCCTGACGGATGGCCTTTT
rrnB T1 terminator Escherichia coli rrnB — 393..480 Rev
DNA Sequence
CAAATAAAACGAAAGGCTCAGTCGGAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT