| 质粒名称: | pCRIS-PITChv2-FBL |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 63672 |
| 存储实验室: | Takashi Yamamoto |
|
载体骨架基本信息 |
|
| 载体名称: | pUC ori vector |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | CRISPR |
| 筛选标记: | 潮霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | EGFP-2A-PuroR |
| 物种来源: | 人工合成 |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | Promoterless |
| 标签/融合蛋白: | -- |
| 克隆方法: | 未知 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | TAGGTCAGGGTGGTCACGAG |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pCRIS-PITChv2-FBL was a gift from Takashi Yamamoto (Addgene plasmid # 63672 ; http://n2t.net/addgene:63672 ; RRID:Addgene_63672) |
| 参考文献部分: | MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T. Nat Protoc. 2016 Jan;11(1):118-33. doi: 10.1038/nprot.2015.140. Epub 2015 Dec 17. 10.1038/nprot.2015.140 PubMed 26678082 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pCRIS-PITChv2-FBL 6033 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION PITCh donor vector for EGFP-2A-PuroR knock-in into human FBL locus.
ACCESSION .
VERSION .
KEYWORDS pCRIS-PITChv2-FBL
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6033)
AUTHORS Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T
TITLE MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the
PITCh systems.
JOURNAL Nat Protoc. 2016 Jan;11(1):118-33. doi: 10.1038/nprot.2015.140. Epub
2015 Dec 17.
PUBMED 26678082
REFERENCE 2 (bases 1 to 6033)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6033
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 86..674
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 575..594
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 828..845
/label=L4440
/note="L4440 vector, forward primer"
primer_bind 943..960
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 944..960
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 1416..1432
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
CDS 1595..2311
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(1640..1661)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(1901..1920)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 2248..2269
/label=EGFP-C
/note="EGFP, forward primer"
CDS 2321..2374
/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 2375..2974
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(2375..2394)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 2871..2891
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
primer_bind complement(3048..3065)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3054..3165
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(3283..3303)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 3288..3617
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 3468..3603
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3530..3549
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3666..4691
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
polyA_signal 4821..4942
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4858..4877)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4912..4931
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
CDS complement(4995..5855)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 5618..5637
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5856..5947)
/gene="bla"
/label=AmpR promoter
primer_bind 5989..6008
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 6014..6033
/label=pENTR-R
/note="pENTR vectors, reverse primer"
ORIGIN
1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
1021 taaaaacccg cttcggcggg tttttttatg gggagagttt agggaaagag catttgtcag
1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc
1441 ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
1501 ggtcattagt tcatagccca tatatggagt tccgcgttac atagcatcgt acgcgtacgt
1561 gtttggttcc tctcacaggc caccccccgg atccatggtg agcaagggcg aggagctgtt
1621 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
1681 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
1741 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
1801 gcagtgcttc agccgttacc ccgaccacat gaagcagcac gacttcttca agtccgccat
1861 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
1921 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
1981 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
2041 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
2101 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
2161 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
2221 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
2281 gatcactctc ggcatggacg agctgtacaa gggaagcgga gagggcagag gaagtctgct
2341 aacatgcggt gacgtcgagg agaatcctgg acctatgacc gagtacaagc ccacggtgcg
2401 cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga
2461 ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct
2521 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
2581 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
2641 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
2701 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
2761 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
2821 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
2881 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
2941 cacctggtgc atgacccgca agcccggtgc ctgaccaagg tgaagaactg aagtccaaac
3001 acgtacgcgt acgatgctct agaatgctga tgggctagca aaatcagcct cgactgtgcc
3061 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
3121 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcacaac actcaaccct
3181 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
3241 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
3301 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
3361 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
3421 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
3481 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
3541 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
3601 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc
3661 acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
3721 tcgacagcgt ttccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
3781 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
3841 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
3901 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
3961 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
4021 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
4081 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
4141 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
4201 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
4261 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
4321 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
4381 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
4441 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
4501 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
4561 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
4621 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
4681 caaaggaata gcacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg
4741 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
4801 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
4861 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
4921 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
4981 cgtaatcatt gtcattacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
5041 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
5101 ttaccatctg gccccagcgc tgcgatgata ccgcgagaac cacgctcacc ggctccggat
5161 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
5221 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
5281 aatagtttgc gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg ctcgtcgttt
5341 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
5401 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
5461 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
5521 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
5581 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
5641 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
5701 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
5761 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
5821 ggaataaggg cgacacggaa atgttgaata ctcatattct tcctttttca atattattga
5881 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
5941 aaacaaatag gggtcagtgt tacaaccaat taaccaattc tgaacattat cgcgagccca
6001 tttatacctg aatatggctc ataacacccc ttg
//
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