| 质粒名称: | pAAVS1-PC-GCaMP6f |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 73503 |
| 存储实验室: | Bruce Conklin |
|
载体骨架基本信息 |
|
| 载体名称: | pAAVS1 |
| 载体出品公司: | Clontech |
| 空载体大小: | 3356bp |
| 完整质粒大小: | 8007bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Puromycin |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | GCaMP6f |
| 物种来源: | Synthetic |
| 插入大小: | 1264 bp |
| 启动子: | CAG |
| 克隆方法: | Restriction Enzyme |
| 5’克隆位点: | SalI (not destroyed) |
| 3’克隆位点: | MluI (not destroyed) |
| 5’测序引物 | CCTCTGCTAACCATGTTCATGC |
| 3’测序引物 | TTCTGATAGGCAGCCTGCAC |
|
文献引用方法 |
|
| 材料和方法部分: |
pAAVS1-PC-GCaMP6f was a gift from Bruce Conklin (Addgene plasmid # 73503 ; http://n2t.net/addgene:73503 ; RRID:Addgene_73503)
|
| 参考文献部分: | CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP, Weissman JS, Qi LS, So PL, Conklin BR. Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi: 10.1016/j.stem.2016.01.022. Epub 2016 Mar 10. 10.1016/j.stem.2016.01.022 PubMed 26971820 |
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-PC-GCaMP6f 8007 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION GCaMP6f knock in construct into the AAVS1 locus.
ACCESSION .
VERSION .
KEYWORDS pAAVS1-PC-GCaMP6f
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8007)
AUTHORS Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen
TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP,
Weissman JS, Qi LS, So PL, Conklin BR
TITLE CRISPR Interference Efficiently Induces Specific and Reversible Gene
Silencing in Human iPSCs.
JOURNAL Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi:
10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
PUBMED 26971820
REFERENCE 2 (bases 1 to 8007)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..8007
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 1..804
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
protein_bind 806..839
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 846..871
/label=SA
/note="splice acceptor site"
CDS 895..948
/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 958..1557
/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(958..977)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 1454..1474
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
primer_bind complement(1589..1606)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1595..1819
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
enhancer 1825..2204
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 2206..2483
/label=chicken beta-actin promoter
intron 2485..3502
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 3426..3448
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 3510..3529
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
regulatory 3562..3571
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3583..3639
/codon_start=1
/product="calmodulin-binding peptide from avian smooth
muscle myosin light chain kinase"
/label=calmodulin-binding peptide
/translation="RRKWNKTGHAVRAIGRLSS"
primer_bind 3852..3873
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(3979..4000)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(4240..4259)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind complement(4908..4927)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 4973..5028
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(5027..5046)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
protein_bind 5377..5410
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 5451..6287
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
rep_origin complement(6335..6923)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6415..6434)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7094..7954)
/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 7717..7736
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
ORIGIN
1 tgctttctct gaccagcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt
61 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
121 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
181 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
241 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
301 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
361 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
421 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
481 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
541 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
601 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
661 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
721 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
781 gtcccctcca ccccacagtg gggcaataac ttcgtatagc atacattata cgaagttata
841 agcttctgac ctcttctctt cctcccacag ggcctcgaga gatctggcag cggagagggc
901 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gctcgagatg
961 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
1021 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
1081 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
1141 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
1201 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
1261 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
1321 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
1381 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
1441 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
1501 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgatct
1561 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
1621 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
1681 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
1741 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
1801 gatgcggtgg gctctatggg tctcgacatt gattattgac tagttattaa tagtaatcaa
1861 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
1921 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
1981 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
2041 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
2101 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
2161 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
2221 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
2281 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg
2341 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
2401 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
2461 aaaagcgaag cgcgcggcgg gcggggagtc gctgcgacgc tgccttcgcc ccgtgccccg
2521 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
2581 gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct
2641 tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg
2701 ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc
2761 cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag
2821 tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg
2881 gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc
2941 ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc
3001 gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg
3061 gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg
3121 gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct
3181 tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc
3241 gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc
3301 cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct
3361 ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag
3421 ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca
3481 tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca
3541 ttttggcaaa gaatgtcgac cgccaccatg gttgactcat cacgtcgtaa gtggaataag
3601 acaggtcacg cagtcagagc tataggtcgg ctgagctcac tcgagaacgt ctatatcaag
3661 gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac
3721 ggcggcgtgc agctcgccta ccactaccag cagaacaccc ccatcggcga cggccccgtg
3781 ctgctgcccg acaaccacta cctgagcgtg cagtccaaac tttcgaaaga ccccaacgag
3841 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
3901 gacgagctgt acaagggcgg taccggaggg agcatggtga gcaagggcga ggagctgttc
3961 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
4021 gtgtccggcg agggtgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
4081 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
4141 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
4201 cccgaaggct acatccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
4261 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
4321 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacct gccggaccaa
4381 ctgactgaag agcagatcgc agaatttaaa gaggaattct ccctatttga caaggacggg
4441 gatgggacaa taacaaccaa ggagctgggg acggtgatgc ggtctctggg gcagaacccc
4501 acagaagcag agctgcagga catgatcaat gaagtagatg ccgacggtga cggcacaatc
4561 gacttccctg agttcctgac aatgatggca agaaaaatga aatacaggga cacggaagaa
4621 gaaattagag aagcgttcgg tgtgtttgat aaggatggca atggctacat cagtgcagca
4681 gagcttcgcc acgtgatgac aaaccttgga gagaagttaa cagatgaaga ggttgatgaa
4741 atgatcaggg aagcagacat cgatggggat ggtcaggtaa actacgaaga gtttgtacaa
4801 atgatgacag cgaagtgaac gcgtgaattc actcctcagg tgcaggctgc ctatcagaag
4861 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct
4921 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
4981 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
5041 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
5101 tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg
5161 aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt
5221 ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat
5281 gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt
5341 ctcttatgga gatccctcga cctgcagccc aagcttataa cttcgtatag catacattat
5401 acgaagttat gcggccgcac cggtatgcat ttaattaaga tatcagcttt actagggaca
5461 ggattggtga cagaaaagcc ccatccttag gcctcctcct tcctagtctc ctgatattgg
5521 gtctaacccc cacctcctgt taggcagatt ccttatctgg tgacacaccc ccatttcctg
5581 gagccatctc tctccttgcc agaacctcta aggtttgctt acgatggagc cagagaggat
5641 cctgggaggg agagcttggc agggggtggg agggaagggg gggatgcgtg acctgcccgg
5701 ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg agctgctctg acgcggctgt
5761 ctggtgcgtt tcactgatcc tggtgctgca gcttccttac acttcccaag aggagaagca
5821 gtttggaaaa acaaaatcag aataagttgg tcctgagttc taactttggc tcttcacctt
5881 tctagtcccc aatttatatt gttcctccgt gcgtcagttt tacctgtgag ataaggccag
5941 tagccagccc cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt ggaaaactcc
6001 ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct actccctttc
6061 tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca tattcctccg
6121 cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt tgagtccttg
6181 gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac catctcatgc
6241 ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctctaag ggcgaattcg
6301 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
6361 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
6421 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
6481 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
6541 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
6601 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
6661 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
6721 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
6781 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
6841 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
6901 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
6961 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
7021 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
7081 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
7141 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
7201 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
7261 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
7321 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
7381 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
7441 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
7501 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
7561 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
7621 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
7681 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
7741 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
7801 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
7861 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
7921 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
7981 gcatttatca gggttgaatt cgccctt
//
