KM-CashGem-Xyl2-URA3-LINEAR

174838 Synthetic Bacterial Expression

Plasmid Information

Purpose

pRS414 backbone containing LINEAR fragment targeting XYL2 in K. marxianus

Depositor

Zengyi Shao

Article

Ploessl et al Nat Chem Biol. 2021 Oct 28. pii: 10.1038

Insert

Codon optimized Cas9:hGem

Tags

hGeminin tag

Promoter

TEF1p

Available Since

Jan. 5, 2022

Availability

Available

Sequence Information

Length

13,387 bp

Topology

circular

Molecule Type

other DNA

View on AddGene.org

Features

CDS 5

Label	Gene	Product	Position	Strand
AmpR	bla	beta-lactamase	759..1620	Rev
DNA Sequence ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA Protein Sequence MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW
TRP1	S. cerevisiae TRP1	phosphoribosylanthranilate isomerase, r…	2804..3479	Fwd
DNA Sequence ATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAG Protein Sequence MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRKRTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDESWQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDWVGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNAKK
Cas9(C)	—	C-terminal portion of Streptococcus pyo…	6396..8781	Fwd
DNA Sequence TGTTTCGACTCTGTCGAAATTTCTGGTGTCGAAGACAGATTCAATGCTTCTTTGGGTACTTACCACGATTTGTTGAAAATCATTAAGGATAAGGATTTCTTAGATAACGAGGAGAACGAAGATATTTTGGAGGATATTGTCTTGACTTTGACTTTGTTTGAGGATAGAGAAATGATCGAGGAAAGATTGAAGACTTACGCTCATTTGTTCGATGATAAGGTCATGAAGCAATTGAAGAGAAGAAGATACACTGGTTGGGGTAGATTGTCCAGAAAGTTGATCAACGGTATTAGAGATAAGCAATCCGGTAAGACCATTTTGGATTTCTTGAAGTCTGACGGTTTCGCTAATAGAAACTTTATGCAATTGATTCACGACGATTCTTTGACTTTTAAGGAAGATATTCAAAAGGCTCAAGTCTCTGGTCAGGGTGATTCTTTGCACGAACACATCGCTAACTTGGCTGGTTCTCCAGCTATCAAAAAAGGTATTTTGCAAACTGTTAAGGTTGTTGACGAATTGGTTAAGGTTATGGGTAGACACAAGCCAGAGAATATTGTCATCGAGATGGCTAGAGAAAATCAAACCACTCAAAAGGGTCAAAAGAACTCCAGAGAGAGAATGAAAAGAATTGAGGAAGGTATCAAGGAGTTGGGTTCCCAAATTTTGAAGGAACACCCAGTCGAGAACACTCAATTGCAAAATGAAAAGTTGTACTTGTATTACTTGCAAAACGGTAGAGATATGTACGTTGACCAAGAATTGGACATTAACAGATTGTCTGACTACGATGTTGATCACATCGTTCCACAATCCTTTTTGAAGGATGACTCTATCGATAACAAGGTTTTGACTAGATCCGACAAGAATAGAGGTAAGTCTGATAACGTTCCATCTGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGTTGTTGAATGCTAAGTTGATTACTCAAAGAAAGTTCGATAACTTGACTAAGGCTGAGAGAGGTGGTTTGTCTGAGTTGGATAAGGCCGGTTTCATTAAGAGACAATTGGTCGAAACCAGACAAATCACCAAGCACGTTGCTCAAATCTTGGACTCTAGAATGAACACCAAGTACGACGAAAACGACAAATTGATTAGAGAGGTTAAGGTCATCACCTTGAAGTCCAAATTGGTTTCCGACTTTAGAAAGGATTTCCAATTCTACAAGGTCAGAGAAATCAATAACTACCACCATGCTCACGACGCCTACTTGAACGCTGTTGTCGGTACTGCTTTGATCAAGAAGTACCCAAAGTTGGAGTCTGAATTTGTCTACGGTGATTACAAAGTTTACGACGTTAGAAAGATGATTGCTAAGTCTGAGCAAGAAATCGGTAAGGCTACTGCTAAATACTTCTTCTACTCCAACATTATGAATTTCTTCAAGACCGAGATTACCTTGGCCAACGGTGAAATCAGAAAGAGACCATTGATTGAAACTAACGGTGAAACCGGTGAGATTGTCTGGGACAAGGGTAGAGATTTCGCTACTGTTAGAAAGGTCTTGTCTATGCCACAAGTCAACATTGTCAAGAAGACCGAAGTCCAGACTGGTGGTTTCTCCAAGGAATCTATTTTGCCAAAGAGAAACTCTGATAAGTTGATTGCCAGAAAGAAAGACTGGGATCCAAAGAAGTACGGTGGTTTCGACTCTCCAACCGTTGCTTACTCCGTTTTGGTTGTCGCTAAGGTTGAAAAGGGTAAGTCCAAGAAGTTGAAGTCCGTCAAGGAGTTGTTGGGTATTACCATCATGGAAAGATCTTCCTTCGAAAAGAACCCAATTGATTTCTTGGAGGCTAAGGGTTACAAGGAAGTCAAGAAGGACTTGATCATTAAGTTGCCAAAGTACTCTTTGTTCGAATTGGAGAACGGTAGAAAGAGAATGTTGGCTTCCGCTGGTGAATTGCAAAAGGGTAACGAATTGGCTTTGCCATCTAAGTACGTTAACTTCTTGTACTTGGCTTCCCACTACGAAAAGTTGAAGGGTTCTCCAGAAGATAACGAACAAAAGCAATTGTTCGTTGAACAACATAAGCACTACTTGGACGAAATCATCGAACAAATCTCTGAATTCTCTAAGAGAGTTATCTTGGCTGACGCTAACTTGGACAAGGTTTTGTCTGCTTACAACAAGCACAGAGACAAGCCAATCAGAGAGCAAGCTGAAAACATCATCCACTTGTTCACTTTGACCAACTTGGGTGCTCCAGCTGCTTTCAAGTACTTCGACACCACCATCGACAGAAAGAGATACACCTCTACCAAGGAAGTTTTGGACGCTACCTTGATCCACCAATCTATCACCGGTTTGTACGAAACCAGAATCGACTTGTCTCAATTGGGTGGTGAC Protein Sequence CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
SV40 NLS	—	nuclear localization signal of SV40 (si…	8793..8814	Fwd
DNA Sequence CCAAAGAAGAAGAGAAAGGTT Protein Sequence PKKKRKV
Gem1 (1-110)	—	degron consisting of residues 1-110 of …	8847..9177	Fwd
DNA Sequence ATGAACCCATCTATGAAGCAAAAGCAAGAAGAAATCAAGGAAAACATCAAGAACTCTTCTGTTCCAAGAAGAACCTTGAAGATGATCCAACCATCTGCTTCTGGTTCTTTGGTTGGTAGAGAAAACGAATTGTCTGCTGGTTTGTCTAAGAGAAAGCACAGAAACGACCACTTGACCTCTACCACCTCTTCTCCAGGTGTTATCGTTCCAGAATCTTCTGAAAACAAGAACTTGGGTGGTGTTACCCAAGAATCTTTCGACTTGATGATCAAGGAAAACCCATCTTCTCAATACTGGAAGGAAGTTGCTGAAAAGAGAAGAAAGGCTTTG Protein Sequence MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELSAGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAEKRRKAL

promoter 6

Label	Gene	Product	Position	Strand
AmpR promoter	bla	—	1620..1725	Rev
DNA Sequence CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT
TRP1 promoter	S. cerevisiae TRP1	—	2523..2804	Fwd
DNA Sequence ACGACATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCATCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGT
T7 promoter	—	—	4203..4222	Fwd
DNA Sequence TAATACGACTCACTATAGG
TEF1 promoter	S. cerevisiae TEF1	—	4247..4646	Fwd
DNA Sequence CCTAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAG
T3 promoter	—	—	12946..12965	Rev
DNA Sequence AATTAACCCTCACTAAAGG
lac promoter	—	—	13033..13064	Rev
DNA Sequence TTTACACTTTATGCTTCCGGCTCGTATGTTG

protein_bind 2

Label	Gene	Product	Position	Strand
lac operator	—	—	13009..13026	Fwd
DNA Sequence TTGTTATCCGCTCACAA
CAP binding site	—	—	13078..13100	Fwd
DNA Sequence ATGAGTGAGCTAACTCACATTA

rep_origin 2

Label	Gene	Product	Position	Strand
ori	—	—	0..589	Rev
DNA Sequence TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
f1 ori	—	—	3581..4037	Rev
DNA Sequence ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTT

terminator 1

Label	Gene	Product	Position	Strand
CYC1 terminator	S. cerevisiae CYC1	—	9192..9440	Fwd
DNA Sequence TCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGC