| 质粒名称: | pcDNA3.1/Puro-CAG-VSFP-CR |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 40257 |
| 存储实验室: | Michael Lin |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1/Puro-CAG |
| 载体出品公司: | Invitrogen/Dr. Paulmurugan Ramasamy |
| 空载体大小: | 6107 bp |
| 完整质粒大小: | 8282 bp |
| 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: | XL10 Gold |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | VSFP-CR |
| 物种来源: | Synthetic; Ciona intestinalis |
| 插入大小: | 2175bp |
| 启动子: | CAG |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | NheI(未破坏) |
| 3’克隆位点: | HindIII(未破坏) |
| 5’测序引物 | AAGGTGGTGGCTGGTGTGGC |
| 3’测序引物 | BGH Reverse |
|
文献引用方法 |
|
| 材料和方法部分: |
pcDNA3.1/Puro-CAG-VSFP-CR was a gift from Michael Lin (Addgene plasmid # 40257 ; http://n2t.net/addgene:40257 ; RRID:Addgene_40257)
|
| 参考文献部分: | Improving FRET dynamic range with bright green and red fluorescent proteins. Lam AJ, St-Pierre F, Gong Y, Marshall JD, Cranfill PJ, Baird MA, McKeown MR, Wiedenmann J, Davidson MW, Schnitzer MJ, Tsien RY, Lin MZ. Nat Methods. 2012 Sep 9. doi: 10.1038/nmeth.2171. 10.1038/nmeth.2171 PubMed 22961245 |
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 pcDNA3.1/Puro-CAG-VSFP-CR 8282 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pcDNA3.1/Puro-CAG-VSFP-CR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8282)
AUTHORS Lam AJ, St-Pierre F, Gong Y, Marshall JD, Cranfill PJ, Baird MA,
McKeown MR, Wiedenmann J, Davidson MW, Schnitzer MJ, Tsien RY, Lin
MZ
TITLE Improving FRET dynamic range with bright green and red fluorescent
proteins.
JOURNAL Nat Methods. 2012 Sep 9. doi: 10.1038/nmeth.2171.
PUBMED 22961245
REFERENCE 2 (bases 1 to 8282)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..8282
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 15..394
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
intron 670..1686
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 1610..1632
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 1694..1713
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
regulatory 1845..1854
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
primer_bind complement(2658..2679)
/label=EGFP-N
/note="EGFP, reverse primer"
CDS 3315..4025
/codon_start=1
/product="monomeric red fluorescent protein derived from
mRuby, with improved photophysical properties (Lam et al.,
2012)"
/label=mRuby2
/translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
AKFAGLGGGMDELYK"
primer_bind complement(4143..4160)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 4149..4373
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 4419..4847
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4506..4525)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4716..4737
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4856..4876)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 4861..5190
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 5041..5176
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 5103..5122
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 5199..5798
/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(5199..5218)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 5695..5715
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
polyA_signal 5958..6079
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5995..6014)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 6049..6068
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(6128..6144)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6128..6144)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(6141..6163)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 6152..6168
/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)."
promoter complement(6176..6206)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6221..6242
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6359..6376)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6530..7115)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6610..6629)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7286..8146)
/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 7909..7928
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(8147..8251)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gacggatcgg ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
61 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
121 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
181 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
241 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
301 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
361 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
421 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
481 attttgtgca gcgatggggg cggggggggg gggcgcgcgc caggcggggc ggggcggggc
541 gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
601 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
661 cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg
721 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
781 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg
841 ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg
901 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc
961 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc
1021 gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
1081 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc
1141 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
1201 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
1261 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg
1321 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
1381 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
1441 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
1501 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
1561 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
1621 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
1681 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc
1741 ctcgaggaat tcactcctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
1801 aatgccctgg ctcacaaata ccactgagat ctgcatgagc tagcgccacc atggagggat
1861 tcgacggttc agattttagt cctccagctg atttagttgg cgttgacggt gcagtcatgc
1921 ggaacgtcgt tgacgtcacg ataaatggtg acgtcactgc tccgccgaaa gctgcgccaa
1981 gaaaaagtga atcggtaaag aaagttcatt ggaatgatgt agaccaagga ccgagtgaaa
2041 aaccagagac aagacaggag gaacgaatag atatacccga gatttcaggt ctatggtggg
2101 gcgagaatga acatggagtg gacgatggga gaatggagat acctactact ggtgtaggtc
2161 gcgtccagtt tcgtgtccga gcagtgattg atcatctagg gatgcgagtc tttggagtct
2221 tcctaatttt cttggacatc atcctcatga tcattgatct cagtcttcca ggaaaaagtg
2281 aatcttcaca atccttttat gacgggatgg ctttggctct ttcttgttat ttcatgctgg
2341 atttaggatt aaggatattt gcctacgggc ccaagaattt cttcaccaac ccctgggagg
2401 ttgctgatgg tttgattatc gttgtcacat tcgtcgtcac gatattttac actgtgttag
2461 atgaatacgt gcaagaaaca ggagccgatg gtttggggca gttggttgtg ttggcccgtt
2521 tgctgcgtgt ggttcgatta gcaagaatat tttattccca ccaacaaatg aaggcttcaa
2581 gcagaagaac aatatcacag aacaagcgtc gaatggtgag caagggcgag gagctgttca
2641 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
2701 tccgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca
2761 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcgtgg
2821 cctgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
2881 ccgaaggcta cgtccaggag cgcaccatct ctttcaagga cgacggtacc tacaagaccc
2941 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
3001 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca
3061 acgtctatat cacggccgac aagcagaaga acggcatcaa ggctaacttc aagatccgcc
3121 acaacgttga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
3181 gcgacggccc cgtgctgctg cccgacaacc actacctgag ccatcagtcc gccctgagca
3241 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtaacc gccgccctcg
3301 agtccggagg cgagatggtg tctaagggcg aagagctgat caaggaaaat atgcgtatga
3361 aggtggtcat ggaaggttcg gtcaacggcc accaattcaa atgcacaggt gaaggagaag
3421 gcaatccgta catgggaact caaaccatga ggatcaaagt catcgaggga ggacccctgc
3481 catttgcctt tgacattctt gccacgtcgt tcatgtatgg cagccgtact tttatcaagt
3541 acccgaaagg cattcctgat ttctttaaac agtcctttcc tgagggtttt acttgggaaa
3601 gagttacgag atacgaagat ggtggagtcg tcaccgtcat gcaggacacc agccttgagg
3661 atggctgtct cgtttaccac gtccaagtca gaggggtaaa ctttccctcc aatggtcccg
3721 tgatgcagaa gaagaccaag ggttgggagc ctaatacaga gatgatgtat ccagcagatg
3781 gtggtctgag gggatacact catatggcac tgaaagttga tggtggtggc catctgtctt
3841 gctctttcgt aacaacttac aggtcaaaaa agaccgtcgg gaacatcaag atgcccggta
3901 tccatgccgt tgatcaccgc ctggaaaggt tagaggaaag tgacaatgaa atgttcgtag
3961 tacaacgcga acacgcagtt gccaagttcg ccgggcttgg tggtgggatg gacgagctgt
4021 acaagtaata aaagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg
4081 cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc
4141 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
4201 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
4261 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
4321 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
4381 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
4441 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
4501 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
4561 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
4621 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
4681 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
4741 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
4801 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
4861 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
4921 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
4981 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
5041 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
5101 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
5161 ggcttttttg gaggcctagg cttttgcaaa aagctcccat gaccgagtac aagcccacgg
5221 tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg
5281 ccgactaccc cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg
5341 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
5401 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
5461 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
5521 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
5581 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
5641 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
5701 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
5761 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgatt cgaaatgacc gaccaagcga
5821 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
5881 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
5941 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
6001 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
6061 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
6121 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
6181 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
6241 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
6301 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
6361 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
6421 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
6481 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
6541 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
6601 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
6661 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
6721 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
6781 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
6841 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
6901 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
6961 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
7021 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca
7081 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
7141 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
7201 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
7261 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
7321 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
7381 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
7441 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
7501 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
7561 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
7621 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
7681 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
7741 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
7801 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
7861 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
7921 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
7981 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
8041 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
8101 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
8161 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
8221 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
8281 tc
//
