| 质粒名称: | pSEED-sfGFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 219639 |
| 存储实验室: | Markus Affolter |
|
载体骨架基本信息 |
|
| 载体名称: | pBluescript KS(+) |
| 载体出品公司: | Clontech |
| 空载体大小: | 4038 bp |
| 完整质粒大小: | 5303bp |
| 载体类型: | 细菌表达 |
| 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: | sfGFP SEED Cassette |
| 插入大小: | 2500bp |
|
文献引用方法 |
|
| 材料和方法部分: | pSEED-sfGFP was a gift from Markus Affolter (Addgene plasmid # 219639 ; http://n2t.net/addgene:219639 ; RRID:Addgene_219639) |
| 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-sfGFP载体质粒基本信息
pSEED-sfGFP质粒图谱载体图谱和pSEED-sfGFP载体序列质粒序列多克隆位点信息
pSEED-sfGFP质粒载体简介
pSEED-sfGFP质粒序列载体序列
LOCUS pSEED-sfGFP 5303 bp DNA circular SYN 14-APR-2025
DEFINITION Permits GoldenGate assembly (BsmBI) of donor plasmids to knock-in
sfGFP using the SEED/Harvest method.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5303)
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 5303)
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-sfGFP
FEATURES Location/Qualifiers
source 1..5303
/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"
promoter 640..658
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
polyA_signal 1371..1492
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1382..1401)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 1436..1455
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
CDS complement(1613..2290)
/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(1675..1698)
/label=DsRed1-C
/note="DsRed1, forward primer"
primer_bind complement(1873..1892)
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 2090..2110
/label=DsRed1-N
/note="DsRed1, reverse primer"
primer_bind 2142..2160
/label=mCherry-R
/note="mCherry, reverse primer"
regulatory 2287..2296
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
primer_bind complement(3131..3150)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(3132..3150)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3157..3174)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(3157..3173)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3166..3188)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 3315..3770
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3402..3421)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3612..3633
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 3796..3900
/gene="bla"
/label=AmpR promoter
CDS join(3901..3969,3970..4761)
/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(4119..4138)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin join(4932..5303,1..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 agcggaagag 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 atgattacgc caagctcgaa attaaccctc actaaaggga
661 acaaaagctg ggtaccgtct cgccggactt gtacagctca tccatgccca gggtgatgcc
721 ggcggcggtc acgaactcca gcagcaccat gtgatcgcgc ttctcgttgg ggtccttgga
781 cagcacgctc tgggtgctca ggtagtggtt atcgggcagc agcactgggc cgtcgccgat
841 gggggtgttc tgctggtagt gatcggccag ctgcacggag ccatcctcca cattgtggcg
901 gatcttgaag ttggccttga tgccgttctt ctgcttatcg gcggtgatgt acacgttgtg
961 gctgttgaag ttgtactcca gcttgtggcc caggatgttg ccatcctcct tgaaatcgat
1021 gcccttcagc tcgatgcggt tcaccagggt atcgccctcg aacttcacct cggcgcgggt
1081 cttgtaggtg ccgtcatcct tgaagctgat ggtgcgctcc tgcacgtagc cctcgggcat
1141 ggcgctcttg aagaaatcgt gctgcttcat gtgatcgggg tagcggctga agcactgcac
1201 gccgtaggtc agggtggtca ccagggtggg ccagggcacg ggcagcttgc cggtggtgca
1261 gatgaacttc agggtcagct tgccgttgga ctagtccgct ggtactgtat ggatgtacgt
1321 cgacgtacat ccatacagta ccagcgggta catccataca gtaccagcgg taagatacat
1381 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
1441 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
1501 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
1561 gtaaaacctc tacaaatgtg gtatggctga ttatgatcta gagtcgcggc cgctacagga
1621 acaggtggtg gcggccctcg gcgcgctcgt actgctccac gatggtgtag tcctcgttgt
1681 gggaggtgat gtccagcttg gagtccacgt agtagtagcc gggcagctgc acgggcttct
1741 tggccatgta gatggacttg aactccacca ggtagtggcc gccgtccttc agcttcaggg
1801 ccttgtggat ctcgcccttc agcacgccgt cgcgggggta caggcgctcg gtggacgcct
1861 cccagcccat agtcttcttc tgcattacgg ggccgtcgga ggggaagttc acgccgatga
1921 acttcacctt gtagatgaag gagccgtcct ggagggagga gtcctgggtc acggtcacca
1981 cgccgccgtc ctcgaagttc atcacgcgct cccacttgaa gccctcgggg aaggacagct
2041 tcttgtagtc ggggatgtcg gcggggtgct tcacgtacac cttggagccg tactggaact
2101 ggggggacag gatgtcccag gcgaagggca gggggccgcc cttggtcacc ttcagcttgg
2161 cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg aactcgtggc
2221 cgttcacgga gccctccatg cgcaccttga agcgcatgaa ctccttgatg acgtcctcgg
2281 aggaggccat ggtggcgacc ggcttcgagc cgattgttta gcttgttcag ctgcgcttgt
2341 ttatttgctt agctttcgct tagcgacgtg ttcactttgc ttgtttgaat tgaattgtcg
2401 ctccgtagac gaagcgcctc tatttatact ccggcggtcg agggttcgaa atcgataagc
2461 ttggatccta attgaattag ctctaattga attagtctct aattgaatta gatccccgcg
2521 ttgttatgat cgtactaccc gcgttgttat gatcgtacta cgctagcgta gtacgatcat
2581 aacaacgcgg ctacctaggg cggatcttga agttggcctt gatgccgttc ttctgcttat
2641 cggcggtgat gtacacgttg tggctgttga agttgtactc cagcttgtgg cccaggatgt
2701 tgccatcctc cttgaaatcg atgcccttca gctcgatgcg gttcaccagg gtatcgccct
2761 cgaacttcac ctcggcgcgg gtcttgtagg tgccgtcatc cttgaagctg atggtgcgct
2821 cctgcacgta gccctcgggc atggcgctct tgaagaaatc gtgctgcttc atgtgatcgg
2881 ggtagcggct gaagcactgc acgccgtagg tcagggtggt caccagggtg ggccagggca
2941 cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgttg gtggcgtcgc
3001 cctcgccctc gccgcgcacg ctgaacttgt ggccgttcac gtcgccatcc agctccacca
3061 ggatgggcac cacgccggtg aacagctcct cgcccttgga caccatagag ccgagacgag
3121 ctccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
3181 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
3241 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
3301 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
3361 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
3421 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
3481 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
3541 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
3601 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
3661 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
3721 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
3781 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
3841 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
3901 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
3961 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
4021 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
4081 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
4141 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
4201 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
4261 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
4321 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
4381 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
4441 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
4501 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
4561 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
4621 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
4681 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
4741 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
4801 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
4861 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
4921 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
4981 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
5041 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
5101 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
5161 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
5221 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
5281 gagcgaacga cctacaccga act
//
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