| 质粒名称: | pAAVS1-P-MCS |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 80488 |
| 存储实验室: | Knut Woltjen |
|
载体骨架基本信息 |
|
| 载体名称: | pBluescript |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 3000 bp |
| 完整质粒大小: | -- |
| 载体修饰: | AAVS1同源臂；无启动子SA-T2A-puro选择盒；MCS |
| 载体类型: | 哺乳动物表达；donor vector (human) |
| 筛选标记: | 嘌呤霉素 |
| 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: | -- |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | -- |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pAAVS1-P-MCS was a gift from Knut Woltjen (Addgene plasmid # 80488 ; http://n2t.net/addgene:80488 ; RRID:Addgene_80488) |
| 参考文献部分: | Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives. Oceguera-Yanez F, Kim SI, Matsumoto T, Tan GW, Xiang L, Hatani T, Kondo T, Ikeya M, Yoshida Y, Inoue H, Woltjen K. Methods. 2015 Dec 18. pii: S1046-2023(15)30181-X. doi: 10.1016/j.ymeth.2015.12.012. 10.1016/j.ymeth.2015.12.012 PubMed 26707206 |
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 pAAVS1-P-MCS 5504 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION donor vector for AAVS1 targeting (puromycin selection) with multiple
cloning site.
ACCESSION .
VERSION .
KEYWORDS pAAVS1-P-MCS
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5504)
AUTHORS Oceguera-Yanez F, Kim SI, Matsumoto T, Tan GW, Xiang L, Hatani T,
Kondo T, Ikeya M, Yoshida Y, Inoue H, Woltjen K
TITLE Engineering the AAVS1 locus for consistent and scalable transgene
expression in human iPSCs and their differentiated derivatives.
JOURNAL Methods. 2015 Dec 18. pii: S1046-2023(15)30181-X. doi:
10.1016/j.ymeth.2015.12.012.
PUBMED 26707206
REFERENCE 2 (bases 1 to 5504)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5504
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 21..125
/gene="bla"
/label=AmpR promoter
CDS 126..986
/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 complement(344..363)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1157..1745
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1646..1665
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 1899..1916
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 2033..2054
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2069..2099
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2107..2123
/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)."
primer_bind 2112..2134
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 2131..2147
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 2131..2147
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
promoter 2168..2186
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
misc_feature 2199..3002
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
misc_feature 3009..3034
/label=SA
/note="splice acceptor site"
CDS 3058..3111
/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 3121..3720
/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(3121..3140)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 3617..3637
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
primer_bind complement(3752..3769)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3758..3982
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
misc_feature 4185..4714
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
primer_bind complement(4860..4879)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(4861..4879)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4886..4903)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(4886..4902)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4895..4917)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 5044..5499
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(5131..5150)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5341..5362
/label=F1ori-F
/note="F1 origin, forward primer"
ORIGIN
1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
2161 agctcgaaat taaccctcac taaagggaac aaaagctgtg ctttctctga ccagcattct
2221 ctcccctggg cctgtgccgc tttctgtctg cagcttgtgg cctgggtcac ctctacggct
2281 ggcccagatc cttccctgcc gcctccttca ggttccgtct tcctccactc cctcttcccc
2341 ttgctctctg ctgtgttgct gcccaaggat gctctttccg gagcacttcc ttctcggcgc
2401 tgcaccacgt gatgtcctct gagcggatcc tccccgtgtc tgggtcctct ccgggcatct
2461 ctcctccctc acccaacccc atgccgtctt cactcgctgg gttccctttt ccttctcctt
2521 ctggggcctg tgccatctct cgtttcttag gatggccttc tccgacggat gtctcccttg
2581 cgtcccgcct ccccttcttg taggcctgca tcatcaccgt ttttctggac aaccccaaag
2641 taccccgtct ccctggcttt agccacctct ccatcctctt gctttctttg cctggacacc
2701 ccgttctcct gtggattcgg gtcacctctc actcctttca tttgggcagc tcccctaccc
2761 cccttacctc tctagtctgt gctagctctt ccagccccct gtcatggcat cttccagggg
2821 tccgagagct cagctagtct tcttcctcca acccgggccc ctatgtccac ttcaggacag
2881 catgtttgct gcctccaggg atcctgtgtc cccgagctgg gaccacctta tattcccagg
2941 gccggttaat gtggctctgg ttctgggtac ttttatctgt cccctccacc ccacagtggg
3001 gcaagcttct gacctcttct cttcctccca cagggcctcg agagatctgg cagcggagag
3061 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc taggctcgag
3121 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
3181 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
3241 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
3301 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
3361 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
3421 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
3481 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
3541 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
3601 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
3661 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
3721 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
3781 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
3841 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
3901 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
3961 ggggatgcgg tgggctctat ggactagtat taattaaatc tagaagtcga cagtactaag
4021 ctttgacaga aaagccccat ccttaggcct cctccttcct agtctcctga tattgggtct
4081 aacccccacc tcctgttagg cagattcctt atctggtgac acacccccat ttcctggagc
4141 catctctctc cttgccagaa cctctaaggt ttgcttacga tggagccaga gaggatcctg
4201 ggagggagag cttggcaggg ggtgggaggg aaggggggga tgcgtgacct gcccggttct
4261 cagtggccac cctgcgctac cctctcccag aacctgagct gctctgacgc ggctgtctgg
4321 tgcgtttcac tgatcctggt gctgcagctt ccttacactt cccaagagga gaagcagttt
4381 ggaaaaacaa aatcagaata agttggtcct gagttctaac tttggctctt cacctttcta
4441 gtccccaatt tatattgttc ctccgtgcgt cagttttacc tgtgagataa ggccagtagc
4501 cagccccgtc ctggcagggc tgtggtgagg aggggggtgt ccgtgtggaa aactcccttt
4561 gtgagaatgg tgcgtcctag gtgttcacca ggtcgtggcc gcctctactc cctttctctt
4621 tctccatcct tctttcctta aagagtcccc agtgctatct gggacatatt cctccgccca
4681 gagcagggtc ccgcttccct aaggccctgc tctgggcttc tgggtttgag tccttggcaa
4741 gcccaggaga ggcgctcagg cttccctgtc ccccttcctc gtccaccatc tcatgcccct
4801 ggctctcctg ccccttccct acaggggttc ctggctctgc tctagcgatc gccaattcgc
4861 cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
4921 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
4981 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
5041 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
5101 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
5161 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
5221 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
5281 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
5341 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
5401 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
5461 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtg
//
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