pTol2-cmlc2-nGFP-nCas9

Plasmid Information

Purpose

Expression of nuclear GFP and nuclear Cas9 specifically in cardiomyocytes driven by the cmlc2(myl7) promoter.

Depositor

Juan Manuel González-Rosa

Article

Keeley et al Cell Rep Methods. 2025 Mar 24;5(3):10100

Insert

Cas9 (Csn1) endonuclease from Streptococcus pyogenes

Sequence Information

Length

10,016 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…	1906..1927	Fwd
DNA Sequence CCAAAGAAGAAGCGTAAGGTA Protein Sequence PKKKRKV
EGFP	—	the original enhanced GFP (Yang et al.,…	1972..2689	Fwd
DNA Sequence ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG Protein Sequence MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
P2A	—	2A peptide from porcine teschovirus-1 p…	2698..2755	Fwd
DNA Sequence GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT Protein Sequence ATNFSLLKQAGDVEENPGP
SV40 NLS	—	nuclear localization signal of SV40 (si…	2767..2788	Fwd
DNA Sequence CCTAAGAAGAAGAGAAAGGTG Protein Sequence PKKKRKV
Cas9	—	Cas9 (Csn1) endonuclease from the Strep…	2794..6895	Fwd
DNA Sequence ATGGATAAGAAGTATAGCATCGGCCTGGATATTGGAACTAACTCCGTGGGTTGGGCAGTGATTACAGACGACTACAAGGTCCCTAGCAAGAAATTTAAGGTGCTGGGTAACACCGACAGGCACAGCATCAAGAAAAATCTGATTGGAGCCCTGCTGTTCGGTTCTGGAGAGACTGCCGAAGCAACACGCCTGAAAAGAACAGCAAGAAGGCGCTATACCAGAAGGAAGAATAGAATCTGTTACCTGCAGGAGATTTTCTCTAACGAAATGGCTAAGGTGGACGATTCATTCTTTCATAGGCTGGAGGAAAGTTTCCTGGTCGAGGAAGATAAGAAACACGAGCGCCATCCTATCTTTGGAAACATTGTGGACGAGGTCGCCTATCACGAAAAATACCCAACCATCTATCATCTGCGCAAGAAACTGGCTGACTCTACTGATAAAGCCGACCTGAGACTGATCTATCTGGCTCTGGCCCACATGATTAAGTTCAGGGGTCATTTTCTGATCGAGGGCGATCTGAACCCCGACAATTCCGATGTGGACAAGCTGTTCATCCAGCTGGTCCAGATTTACAATCAGCTGTTTGAGGAAAACCCTATTAATGCTTCCAGAGTGGACGCAAAAGCTATCCTGTCAGCCAGGCTGTCCAAGTCACGCAGACTGGAGAACCTGATTGCACAGCTGCCCGGAGAAAAGAGGAACGGTCTGTTTGGAAATCTGATCGCTCTGAGTCTGGGCCTGACTCCTAACTTCAAAAGCAATTTTGATCTGGCTGAGGACGCCAAACTGCAGCTGTCAAAGGACACATATGACGATGACCTGGATAACCTGCTGGCACAGATCGGAGATCAGTACGCTGACCTGTTCCTGGCTGCCAAAAATCTGTCCGACGCAATCCTGCTGTCAGATATTCTGAGAGTGAACAGCGAGATTACAAAAGCTCCTCTGAGTGCCAGCATGATCAAGAGATATGACGAGCACCATCAGGATCTGACCCTGCTGAAGGCTCTGGTCAGGCAGCAGCTGCCAGAGAAGTACAAGGAAATTTTCTTTGATCAGTCCAAGAACGGCTACGCCGGTTATATCGACGGAGGCGCATCACAGGAGGAATTCTACAAGTTTATCAAACCTATTCTGGAGAAGATGGACGGAACTGAGGAACTGCTGGTGAAACTGAATAGAGAGGACCTGCTGAGGAAGCAGCGCACATTTGATAACGGTTCCATCCCACACCAGATTCATCTGGGAGAGCTGCACGCTATCCTGAGGCGCCAGGAAGACTTCTACCCCTTTCTGAAAGATAACCGCGAGAAGATCGAAAAAATTCTGACCTTCAGAATCCCTTACTATGTGGGTCCACTGGCTCGCGGAAACAGCAGATTTGCCTGGATGACTCGCAAATCCGAGGAAACCATTACTCCTTGGAACTTCGAGGAAGTGGTCGATAAGGGCGCCTCTGCACAGTCCTTCATCGAGAGAATGACTAATTTTGACAAAAACCTGCCCAATGAGAAAGTGCTGCCTAAGCACTCCCTGCTGTACGAGTATTTCACTGTCTATAACGAACTGACAAAGGTGAAATACGTCACCGAGGGCATGAGAAAGCCAGCCTTCCTGTCAGGAGAGCAGAAGAAAGCAATCGTGGATCTGCTGTTTAAAACCAACAGGAAAGTGACTGTCAAGCAGCTGAAGGAGGACTACTTCAAGAAAATTGAATGCTTCGATTCCGTGGAGATCAGCGGAGTCGAAGACAGATTTAACGCAAGCCTGGGCGCTTACCACGATCTGCTGAAGATCATTAAGGATAAAGACTTCCTGGACAACGAGGAAAATGAGGATATCCTGGAAGACATTGTGCTGACACTGACCCTGTTTGAGGACAGAGGAATGATCGAGGAAAGACTGAAAACCTATGCTCATCTGTTCGATGACAAGGTGATGAAACAGCTGAAGAGAAGGCGCTACACTGGCTGGGGTAGACTGAGCAGGAAGCTGATCAACGGCATTAGGGATAAACAGTCAGGAAAGACAATCCTGGACTTTCTGAAAAGTGATGGCTTCGCCAACCGCAATTTTATGCAGCTGATTCACGATGACAGTCTGACCTTCAAAGAGGACATCCAGAAGGCTCAGGTGTCTGGACAGGGCCACTCCCTGCATGAGCAGATTGCAAACCTGGCTGGAAGCCCAGCCATCAAGAAAGGCATTCTGCAGACAGTGAAAATCGTCGATGAGCTGGTGAAAGTCATGGGCCATAAGCCCGAAAACATCGTGATTGAGATGGCTCGCGAAAATCAGACAACCCAGAAGGGTCAGAAGAACAGTAGAGAGAGGATGAAAAGAATCGAGGAAGGCATTAAGGAGCTGGGTAGCCAGATCCTGAAAGAGCACCCAGTGGAAAACACACAGCTGCAGAATGAGAAGCTGTATCTGTACTATCTGCAGAATGGAAGAGATATGTACGTGGACCAGGAGCTGGATATTAACAGGCTGTCTGATTACGACGTGGATCATATCGTCCCCCAGAGTTTCATCAAAGATGACAGCATTGACAACAAGGTGCTGACCAGGTCCGACAAAAACAGAGGAAAATCAGATAATGTCCCTAGTGAGGAAGTGGTCAAGAAAATGAAGAACTACTGGAGACAGCTGCTGAATGCCAAACTGATCACTCAGAGGAAGTTTGATAACCTGACAAAAGCAGAGCGCGGTGGACTGTCAGAACTGGACAAAGCTGGATTCATCAAGAGGCAGCTGGTGGAAACACGCCAGATCACTAAACACGTCGCACAGATTCTGGATAGTCGCATGAACACAAAGTACGATGAGAATGACAAACTGATCAGAGAAGTGAAGGTCATTACCCTGAAGAGTAAACTGGTCAGCGACTTTAGGAAAGATTTCCAGTTTTATAAGGTCCGCGAGATTAACAATTATCACCATGCCCATGACGCATACCTGAACGCCGTGGTCGGTACCGCACTGATCAAGAAATACCCAAAACTGGAGAGCGAATTCGTGTACGGAGACTATAAGGTGTACGATGTCAGAAAAATGATCGCCAAGTCCGAGCAGGAAATTGGAAAAGCTACTGCCAAGTATTTCTTTTACTCAAACATCATGAATTTCTTTAAGACAGAGATCACCCTGGCCAATGGAGAAATCCGCAAAAGGCCCCTGATTGAGACAAACGGAGAGACAGGCGAAATCGTGTGGGACAAAGGCAGAGATTTTGCAACCGTGAGGAAGGTCCTGAGCATGCCTCAAGTGAATATCGTCAAGAAAACTGAGGTGCAGACAGGCGGTTTCTCAAAAGAAAGTATTCTGCCAAAACGCAACTCTGATAAGCTGATCGCTAGAAAGAAAGACTGGGACCCTAAGAAGTATGGAGGCTTTGACTCTCCCACTGTGGCATACTCCGTCCTGGTGGTCGCTAAGGTGGAGAAGGGCAAAAGCAAGAAACTGAAATCTGTCAAGGAGCTGCTGGGTATCACAATTATGGAGAGAAGCTCTTTCGAGAAGAACCCAATCGATTTTCTGGAGGCCAAAGGTTATAAGGAAGTGAAGAAAGACCTGATCATTAAACTGCCCAAGTACAGTCTGTTTGAGCTGGAAAACGGCAGGAAACGCATGCTGGCAAGCGCTGGAGAGCTGCAGAAAGGCAATGAACTGGCCCTGCCTTCTAAGTACGTGAACTTCCTGTATCTGGCAAGCCACTACGAGAAGCTGAAAGGATCTCCAGAGGATAACGAACAGAAACAGCTGTTTGTGGAGCAGCACAAGCATTATCTGGACGAGATCATTGAACAGATTAGCGAGTTCTCTAAAAGAGTGATCCTGGCCGACGCAAATCTGGATAAGGTCCTGTCTGCTTACAACAAACACAGAGATAAGCCCATCAGGGAGCAGGCCGAAAATATCATTCATCTGTTCACTCTGACAAACCTGGGCGCACCTGCAGCTTTCAAGTACTTCGACACTACAATCGATAGAAAGAGGTACACCTCCACTAAGGAGGTGCTGGACGCTACACTGATCCATCAGAGTATTACCGGCCTGTACGAAACAAGGATTGACCTGTCTCAGCTGGGTGGCGAC Protein Sequence MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
SV40 NLS	—	nuclear localization signal of SV40 (si…	6910..6931	Fwd
DNA Sequence CCTAAGAAGAAGAGAAAGGTG Protein Sequence PKKKRKV
AmpR	bla	beta-lactamase	9031..9892	Rev
DNA Sequence ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCACTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAACCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA Protein Sequence MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGTGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW

RBS 1

Label	Gene	Product	Position	Strand
Shine-Dalgarno sequence	—	—	6807..6816	Fwd
DNA Sequence TAAGGAGGT

polyA_signal 1

Label	Gene	Product	Position	Strand
SV40 poly(A) signal	—	—	6972..7107	Fwd
DNA Sequence GATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT

promoter 3

Label	Gene	Product	Position	Strand
T3 promoter	—	—	7831..7850	Rev
DNA Sequence AATTAACCCTCACTAAAGG
lac promoter	—	—	7918..7949	Rev
DNA Sequence TTTACACTTTATGCTTCCGGCTCGTATGTTG
AmpR promoter	bla	—	9892..9997	Rev
DNA Sequence CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT

protein_bind 2

Label	Gene	Product	Position	Strand
lac operator	—	—	7894..7911	Fwd
DNA Sequence TTGTTATCCGCTCACAA
CAP binding site	—	—	7963..7985	Fwd
DNA Sequence ATGAGTGAGCTAACTCACATTA

rep_origin 1

Label	Gene	Product	Position	Strand
ori	—	—	8272..8861	Rev
DNA Sequence TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA