| 质粒名称: | pSEED |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 219658 |
| 存储实验室: | Markus Affolter |
|
载体骨架基本信息 |
|
| 载体名称: | pUC19-Kan |
| 载体出品公司: | Clontech |
| 空载体大小: | 3975bp |
| 完整质粒大小: | 3975bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | Empty SEED Cassette (3xP3-dsRed) |
| 插入大小: | 1300bp |
| 3’测序引物 | SV40pA-R |
|
文献引用方法 |
|
| 材料和方法部分: | pSEED was a gift from Markus Affolter (Addgene plasmid # 219658 ; http://n2t.net/addgene:219658 ; RRID:Addgene_219658) |
| 参考文献部分: | Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing. Aguilar G, Bauer M, Vigano MA, Schnider ST, Brugger L, Jimenez-Jimenez C, Guerrero I, Affolter M. Dev Cell. 2024 Oct 7;59(19):2672-2686.e5. doi: 10.1016/j.devcel.2024.06.004. Epub 2024 Jul 5. 10.1016/j.devcel.2024.06.004 PubMed 38971155 |
买家导航
pSEED载体质粒基本信息
pSEED质粒图谱载体图谱和pSEED载体序列质粒序列多克隆位点信息
pSEED质粒载体简介
pSEED质粒序列载体序列
LOCUS pSEED 3975 bp DNA circular SYN 14-APR-2025
DEFINITION Permits GoldenGate assembly of new SEED cassettes via Golden Gate
assembly (BsaI).
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3975)
AUTHORS Aguilar G, Bauer M, Vigano MA, Schnider ST, Brugger L,
Jimenez-Jimenez C, Guerrero I, Affolter M
TITLE Seamless knockins in Drosophila via CRISPR-triggered single-strand
annealing.
JOURNAL Dev Cell. 2024 Oct 7;59(19):2672-2686.e5. doi:
10.1016/j.devcel.2024.06.004. Epub 2024 Jul 5.
PUBMED 38971155
REFERENCE 2 (bases 1 to 3975)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 14, 2025 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.devcel.2024.06.004"; journalName: "Dev Cell"; date:
"2024-10-7- 7"; volume: "59"; issue: "19"; pages: "2672-2686.e5"
COMMENT Sequence Label: pSEED
FEATURES Location/Qualifiers
source 1..3975
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 118..137
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 371..388
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 505..526
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter join(541..546,547..564,565..571)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 579..595
/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 584..606
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 603..619
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 603..619
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
polyA_signal 755..876
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(766..785)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 820..839
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
CDS complement(997..1674)
/codon_start=1
/product="rapidly maturing tetrameric variant of DsRed
fluorescent protein (Bevis and Glick, 2002)"
/label=DsRed-Express
/note="mammalian codon-optimized"
/translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
LFL"
primer_bind complement(1059..1082)
/label=DsRed1-C
/note="DsRed1, forward primer"
primer_bind complement(1257..1276)
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 1474..1494
/label=DsRed1-N
/note="DsRed1, reverse primer"
primer_bind 1526..1544
/label=mCherry-R
/note="mCherry, reverse primer"
regulatory 1671..1680
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
primer_bind complement(2023..2040)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2023..2039)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2032..2054)
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind complement(2248..2267)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2367..2389
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2427..2445)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2513..2617
/gene="bla"
/label=AmpR promoter
CDS 2618..3433
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRFAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind complement(2691..2710)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
rep_origin join(3604..3975,1..217)
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
361 gaccgagcgc agcgagtcag tgagcgagga agcggaacag cgcccaatac gcaaaccgcc
421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
601 cacaggaaac agctatgacc atgctcgagc caagctcggc gcgcccgtct cgccggaacc
661 gagaccggtc tctactagtc cgctggtact gtatggatgt acgtcgacgt acatccatac
721 agtaccagcg ggtacatcca tacagtacca gcggtaagat acattgatga gtttggacaa
781 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
841 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
901 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa
961 tgtggtatgg ctgattatga tctagagtcg cggccgctac aggaacaggt ggtggcggcc
1021 ctcggcgcgc tcgtactgct ccacgatggt gtagtcctcg ttgtgggagg tgatgtccag
1081 cttggagtcc acgtagtagt agccgggcag ctgcacgggc ttcttggcca tgtagatgga
1141 cttgaactcc accaggtagt ggccgccgtc cttcagcttc agggccttgt ggatctcgcc
1201 cttcagcacg ccgtcgcggg ggtacaggcg ctcggtggac gcctcccagc ccatagtctt
1261 cttctgcatt acggggccgt cggaggggaa gttcacgccg atgaacttca ccttgtagat
1321 gaaggagccg tcctggaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa
1381 gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcttgt agtcggggat
1441 gtcggcgggg tgcttcacgt acaccttgga gccgtactgg aactgggggg acaggatgtc
1501 ccaggcgaag ggcagggggc cgcccttggt caccttcagc ttggcggtct gggtgccctc
1561 gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc
1621 catgcgcacc ttgaagcgca tgaactcctt gatgacgtcc tcggaggagg ccatggtggc
1681 gaccggcttc gagccgattg tttagcttgt tcagctgcgc ttgtttattt gcttagcttt
1741 cgcttagcga cgtgttcact ttgcttgttt gaattgaatt gtcgctccgt agacgaagcg
1801 cctctattta tactccggcg gtcgagggtt cgaaatcgat aagcttggat cctaattgaa
1861 ttagctctaa ttgaattagt ctctaattga attagatccc cgcgttgtta tgatcgtact
1921 acccgcgttg ttatgatcgt actacgctag cgtagtacga tcataacaac gcggctacct
1981 agggtagaga ccggtctcag agccgagacg acgcgtcaat tcactggccg tcgttttaca
2041 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
2101 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
2161 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
2221 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
2281 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
2341 cgcttacaga caagctgtga cagtctccgg gagctgcatg tgtcagaggt tttcaccgtc
2401 atcaccgaaa cgcgcgagac aaaagggcct cgtgatacgc ctatttttat aggttaatgt
2461 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
2521 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
2581 ctgataaatg cttcaataat attgaaaaag gaagagtatg agccatattc aacgggaaac
2641 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
2701 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
2761 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
2821 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
2881 ccgtactcct gatgatgcat ggttactcac cactgcgatc cctgggaaaa cagcattcca
2941 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
3001 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
3061 tttcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
3121 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
3181 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
3241 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
3301 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
3361 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
3421 tgagtttttc taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
3481 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
3541 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
3601 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
3661 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
3721 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
3781 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
3841 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
3901 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
3961 gacctacacc gaact
//
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