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- 详细信息
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
Plasmid#85434
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：pHR_Gal4UAS_Pembrolizumab_heavychain_T2A_ Pembrolizumab_lightchain_PGK_mCherry哺乳抗体质粒
别称: Plasmid#85434
质粒属性
质粒简介
pHR_Gal4UAS_Pembrolizumab_heavychain_T2A_ Pembrolizumab_lightchain_PGK_mCherry是一个哺乳细胞抗体表达质粒。
质粒图谱
质粒序列
LOCUS Exported 12264 bp ds-DNA circular SYN 03-JAN-2016
FEATURES Location/Qualifiers
source 1..12264
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 96..7517
/label=match to pHR-CMVLacZ
/note="match to pHR-CMVLacZ"
/note="color: #a6acb3"
primer_bind complement(320..337)
/label=SP6
/note="SP6"
/note="color: #00ff00; direction: LEFT"
CDS 1231..2091
/codon_start=1
/label=bla (AmpR)
/note="bla"
/note="color: #993366"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
CDS 1429..2088
/label=AmpR
/note="AmpR"
/note="color: #ffff00; direction: RIGHT"
rep_origin 2186..2868
/label=ColE1 origin
/note="ColE1 origin"
/note="color: #808080"
promoter 3084..3410
/label=SV40 - Invitrogen
/note="SV40 - Invitrogen promoter"
/note="color: #0000ff"
promoter 3229..3425
/label=SV40 early promoter
/note="SV40 early promoter"
/note="color: #6495ed"
polyA_site 5105..5123
/label=SV40 pA
/note="SV40 pA"
/note="color: #ccff66"
polyA_signal complement(5138..5329)
/label=SV40 late polyA
/note="SV40 late polyA"
/note="color: #ffc0cb; direction: LEFT"
polyA_site 5199..5330
/label=SV40 early poly A
/note="SV40 early poly A"
/note="color: #80ff00"
polyA_site 5199..5329
/label=SV40 early poly A (2)
/note="SV40 early poly A"
/note="color: #66ff66"
repeat_region 7518..7697
/label=cPPT
/note="cPPT"
/note="color: #a6acb3"
primer_bind 7577..7598
/note="QB2741"
/note="color: #ff0000; direction: RIGHT"
primer_bind 7673..7696
/note="cPPT_5'seq"
/note="color: #a6acb3; direction: RIGHT"
misc_feature 7716..7721
/note="EcoRI"
/note="color: #a6acb3"
misc_feature 7722..7807
/note="Gal4"
/note="color: #a6acb3"
misc_feature 7828..7847
/note="Gal4"
/note="color: #a6acb3"
misc_feature 7848..7853
/note="SpeI"
/note="color: #a6acb3"
promoter 7854..7971
/note="mCMV"
/note="color: #ffffff"
misc_feature 7972..7991
/note="20BPhomo BamHI-pHR Gal4UAS synNotch_fwd"
/note="color: #a6acb3"
misc_feature 7992..9445
/note="Pembrolizumab_HC_gBlock"
/note="color: #a6acb3"
misc_feature 7992..7997
/note="Kozak Sequence"
/note="color: #ff00ff"
misc_feature 7998..8054
/note="Human IgG Heavy Chain Signal Peptide"
/note="Kober et al 2013 Biotech and Bioeng
Codon Optimized"
/note="color: #ff0000"
misc_feature 8055..8084
/note="myc tag"
/note="label=myc tag - ApEinfo_fwdcolor=#ffff80 -
ApEinfo_revcolor=green - ApEinfo_graphicformat=arrow_data
{{0 1 2 0 0 -1} {} 0} - width 5 offset 0 - 165..194"
/note="color: #dc143c; direction: RIGHT"
misc_feature 8055..8084
/note="cw35"
/note="label=cw35 - ApEinfo_fwdcolor=#8080c0 -
ApEinfo_revcolor=green - ApEinfo_graphicformat=arrow_data
{{0 1 2 0 0 -1} {} 0} - width 5 offset 0 - 165..214"
/note="color: #dc143c; direction: RIGHT"
misc_feature complement(8055..8084)
/note="cw34"
/note="label=cw34 - ApEinfo_fwdcolor=cyan -
ApEinfo_revcolor=green - ApEinfo_graphicformat=arrow_data
{{0 1 2 0 0 -1} {} 0} - width 5 offset 0 -
complement(145..194)"
/note="color: #00ffff; direction: LEFT"
misc_feature 8055..8084
/product="Myc (human c-Myc oncogene) epitope tag"
/note="Myc"
/note="color: #cc99b2"
misc_feature 8085..9425
/note="Pembrolizumab Heavy Chain"
/note="IDT Codon Optimized (gBlocks setting, homo sapiens)
From AA :
http://www.drugbank.ca/drugs/DB09037/polypeptide_sequences.
fasta"
/note="color: #00ff00"
misc_feature 8085..8444
/note="Pembrolizumab HC Variable"
/note="color: #0000ff"
misc_feature 8445..9425
/note="huIgG4 Constant"
/note=" (http://www.uniprot.org/uniprot/P01861)"
/note="color: #00ffff"
misc_feature 8766..8768
/note="S -> P"
/note="color: #993366"
misc_feature 9426..10244
/note="Pembrolizumab_LC_gBlock"
/note="color: #a6acb3"
misc_feature 9426..9560
/note="mAb_Center_gBlock"
/note="color: #ffcc99"
misc_feature 9426..9437
/note="Furin Cleavage Site"
/note="color: #ff0000"
misc_feature 9438..9449
/note="SGSG Spacer"
/note="color: #a6acb3"
misc_feature 9450..9503
/note="T2A"
/note="Codon optimized from AA sequence from Yang et al.
2008"
/note="color: #ffcc00"
misc_feature 9504..9560
/note="Human IgG Light Chain Signal Peptide"
/note="Kober et al 2013 Biotech and Bioeng
Codon Optimized"
/note="color: #ff0000"
misc_feature 9561..9587
/note="HA Tag"
/note="Sequence from Wiki"
/note="color: #ff99cc"
misc_feature 9588..10241
/note="Pembrolizumab Light Chain"
/note="IDT Codon Optimized (gBlock setting, homo sapien)
From AA :
http://www.drugbank.ca/drugs/DB09037/polypeptide_sequences.
fasta"
/note="color: #99cc00"
misc_feature 9588..9923
/note="Pembrolizumab LC Variable"
/note="color: #ff99cc"
misc_feature 9924..10241
/note="Ig kappa chain C region"
/note="color: #ccffcc"
misc_feature 10367..10866
/note="promPGK"
/note="color: #996633; direction: RIGHT"
misc_feature 10887..11597
/note="mCherry"
/note="color: #ff0000; direction: RIGHT"
enhancer 11673..12264
/label=WPRE
/note="WPRE"
/note="color: #a6acb3"
ORIGIN
1 cgataccgtc gaccaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
61 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
121 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
181 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
241 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatct
301 agaattaatt ccgtgtattc tatagtgtca cctaaatcgt atgtgtatga tacataaggt
361 tatgtattaa ttgtagccgc gttctaacga caatatgtac aagcctaatt gtgtagcatc
421 tggcttactg aagcagaccc tatcatctct ctcgtaaact gccgtcagag tcggtttggt
481 tggacgaacc ttctgagttt ctggtaacgc cgtcccgcac ccggaaatgg tcagcgaacc
541 aatcagcagg gtcatcgcta gccagatcct ctacgccgga cgcatcgtgg ccggcatcac
601 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
661 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
721 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
781 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
841 tcgaatggtg cactctcagt acaatctagc tctgatgccg catagttaag ccagccccga
901 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
961 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
1021 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
1081 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
1141 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
1201 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
1261 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
1321 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
1381 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
1441 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
1501 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
1561 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
1621 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
1681 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
1741 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
1801 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
1861 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
1921 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
1981 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
2041 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
2101 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
2161 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
2221 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
2281 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
2341 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
2401 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
2461 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
2521 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
2581 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
2641 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
2701 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
2761 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
2821 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
2881 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
2941 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
3001 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
3061 gcgcgttggc cgattcatta atgcagctgt ggaatgtgtg tcagttaggg tgtggaaagt
3121 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
3181 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
3241 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
3301 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
3361 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
3421 gcaaaaagct tggacacaag acaggcttgc gagatatgtt tgagaatacc actttatccc
3481 gcgtcaggga gaggcagtgc gtaaaaagac gcggactcat gtgaaatact ggtttttagt
3541 gcgccagatc tctataatct cgcgcaacct attttcccct cgaacacttt ttaagccgta
3601 gataaacagg ctgggacact tcacatgagc gaaaaataca tcgtcacctg ggacatgttg
3661 cagatccatg cacgtaaact cgcaagccga ctgatgcctt ctgaacaatg gaaaggcatt
3721 attgccgtaa gccgtggcgg tctgtaccgg gtgcgttact ggcgcgtgaa ctgggtattc
3781 gtcatgtcga taccgtttgt atttccagct acgatcacga caaccagcgc gagcttaaag
3841 tgctgaaacg cgcagaaggc gatggcgaag gcttcatcgt tattgatgac ctggtggata
3901 ccggtggtac tgcggttgcg attcgtgaaa tgtatccaaa agcgcacttt gtcaccatct
3961 tcgcaaaacc ggctggtcgt ccgctggttg atgactatgt tgttgatatc ccgcaagata
4021 cctggattga acagccgtgg gatatgggcg tcgtattcgt cccgccaatc tccggtcgct
4081 aatcttttca acgcctggca ctgccgggcg ttgttctttt taacttcagg cgggttacaa
4141 tagtttccag taagtattct ggaggctgca tccatgacac aggcaaacct gagcgaaacc
4201 ctgttcaaac cccgctttaa acatcctgaa acctcgacgc tagtccgccg ctttaatcac
4261 ggcgcacaac cgcctgtgca gtcggccctt gatggtaaaa ccatccctca ctggtatcgc
4321 atgattaacc gtctgatgtg gatctggcgc ggcattgacc cacgcgaaat cctcgacgtc
4381 caggcacgta ttgtgatgag cgatgccgaa cgtaccgacg atgatttata cgatacggtg
4441 attggctacc gtggcggcaa ctggatttat gagtgggccc cggatctttg tgaaggaacc
4501 ttacttctgt ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt
4561 aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt
4621 ttagattcca acctatggaa ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga
4681 aaacctgttt tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgactctca
4741 acattctact cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga
4801 attgctaagt tttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat
4861 ttacaccaca aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatattctgt
4921 aacctttata agtaggcata acagttataa tcataacata ctgttttttc ttactccaca
4981 caggcataga gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt
5041 aatttgtaaa ggggttaata aggaatattt gatgtatagt gccttgacta gagatcataa
5101 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
5161 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
5221 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
5281 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga
5341 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca
5401 attacctagt ggtttcattt actctaaacc tgtgattcct ctgaattatt ttcattttaa
5461 agaaattgta tttgttaaat atgtactaca aacttagtag ttggaagggc taattcactc
5521 ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
5581 ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta
5641 caagctagta ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag
5701 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg
5761 gaggtttgac agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt
5821 caagaactgc tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag
5881 gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc
5941 tttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
6001 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
6061 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
6121 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
6181 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
6241 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
6301 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
6361 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
6421 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
6481 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
6541 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
6601 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg gtgatcttca
6661 gacctggacg atatatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
6721 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
6781 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
6841 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
6901 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
6961 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
7021 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
7081 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
7141 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
7201 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
7261 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
7321 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
7381 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
7441 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
7501 gaacggatct cgacggtcgc caaatggcag tattcatcca caattttaaa agaaaagggg
7561 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
7621 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
7681 gcagagatcc agtttggatc gataagcttg atatcgaatt cggagcactg tcctccgaac
7741 gtcggagcac tgtcctccga acgtcggagc actgtcctcc gaacgtcgga gcactgtcct
7801 ccgaacggag catgtcctcc gaacgtcgga gcactgtcct ccgaacgact agttaggcgt
7861 gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
7921 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctctcgaca
7981 ttcgttggat cgccgccatg gattggacat ggagagtgtt ttgtctgctt gcggtgacac
8041 ctggcgctca tcctgagcag aagctgatca gcgaggagga cctgcaggta caactggtcc
8101 agtcaggcgt cgaggttaaa aagcccggtg cctcagtgaa ggtctcctgc aaagccagtg
8161 gttacacctt cactaattac tatatgtatt gggtcagaca ggcacccggc cagggtcttg
8221 aatggatggg aggcatcaat cccagcaatg gagggacaaa cttcaatgaa aagttcaaaa
8281 atcgcgtgac cctcacaacc gactcctcca caacaacagc atatatggaa ctgaagtctc
8341 ttcagtttga tgacaccgcc gtctactact gcgcccgacg agactaccgg ttcgatatgg
8401 gattcgacta ttggggtcag gggacgacag tgacagtctc ctccgcatct acaaagggac
8461 catcagtgtt tcctctggcc ccgtgctccc gctctaccag cgaatctacc gcagcattgg
8521 gctgtttggt gaaggactac ttccctgagc cagtgaccgt gtcctggaac agcggggcac
8581 tcactagcgg agtgcacaca tttccagccg tgcttcaaag cagcggtctc tattccctct
8641 ccagcgtggt gactgtgcca tccagctctc tcggcacgaa gacctatacc tgcaacgtgg
8701 accacaagcc ttcaaatacc aaggtggata aacgcgtcga atccaaatat ggcccaccct
8761 gcccgccatg tccagctcca gagtttctcg gcgggcctag cgtgttcttg tttcccccca
8821 aacccaagga cacgctcatg atttccagaa cacctgaagt tacctgcgtc gtcgtagacg
8881 tcagtcagga ggacccggaa gttcagttta actggtacgt ggacggtgtt gaggtgcata
8941 atgccaaaac caagccaaga gaagagcagt tcaattctac atatcgagtg gtgtcagtcc
9001 tgacagttct ccaccaggac tggttgaacg gcaaagagta caaatgcaag gtctccaaca
9061 aaggattgcc gagcagtata gagaaaacaa ttagtaaagc taagggtcag cccagagagc
9121 cccaggtgta cacgttgcca ccttcacagg aagagatgac gaagaaccaa gtgtctctga
9181 cctgcctggt gaagggcttc tacccatcag acattgcagt ggagtgggaa agcaatgggc
9241 agcctgagaa caactacaaa actacacccc ccgtcctgga cagcgatggc agtttcttcc
9301 tgtactcaag gttgactgtg gacaaaagca gatggcagga aggcaatgtg ttcagctgta
9361 gtgtgatgca cgaggccctc cacaatcatt acactcagaa atccctctct ctgagcctgg
9421 gaaagaggaa gcgacggagt gggtcaggcg aggggagagg ctcactcctc acgtgtggag
9481 acgttgaaga aaaccccgga ccaatggctt ggagtcctct gttcctgacc ctgattactc
9541 actgcgctgg aagctgggct tacccatacg atgttccaga ttacgctgag attgttctga
9601 cccaatctcc tgcaacactg agcctgtccc ccggagagag ggcaaccctc tcctgccgcg
9661 ccagtaaggg agtgtctacg tctggatatt cctatctcca ttggtatcag cagaagcctg
9721 gccaggcgcc caggcttctc atctaccttg cttcctatct ggagtccggg gtgccagccc
9781 gcttttctgg gtccggctct ggtaccgact tcaccctgac aatttcctca ctggagcctg
9841 aggatttcgc tgtctactat tgtcaacatt ccagagacct tcccctgacc tttggagggg
9901 gtacaaaagt ggagataaaa cgaacagtgg ccgccccatc tgtattcatc tttcctccat
9961 ctgatgaaca gcttaagtcc ggcactgctt ctgttgtttg cctgcttaac aatttttacc
10021 cgcgggaagc caaggtgcaa tggaaagtgg acaacgccct gcagtctggt aacagtcagg
10081 aaagcgtgac tgaacaggac agcaaagact ccacatactc cttgagttcc accttgacgc
10141 tcagtaaggc agactacgag aagcacaagg tttatgcctg tgaagtgact caccagggcc
10201 tctctagccc agtgactaag tccttcaacc gcggggagtg ttgagatcct tgacttgcgg
10261 ccgcaactcc cacctgcaac atgcgtgact gactgaggcc gcgactctag agtcgacctg
10321 catctaggcg ccggaattag atctctcgag gttaacgaat tctaccgggt aggggaggcg
10381 cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac
10441 acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg
10501 ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc
10561 gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc
10621 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa
10681 tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc
10741 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
10801 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc
10861 ctttcgacct gcagcccaag cttaccatgg tgagcaaggg cgaggaggat aacatggcca
10921 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
10981 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
11041 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
11101 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
11161 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
11221 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
11281 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
11341 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
11401 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
11461 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg
11521 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
11581 acgagctgta caagtaaagc ggccgcgact ctagagtcga cctgcaggca tgcaagcttg
11641 caggcatgca agcttgatat caagcttatc gataatcaac ctctggatta caaaatttgt
11701 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
11761 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
11821 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
11881 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
11941 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
12001 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
12061 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
12121 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
12181 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
12241 tccctttggg ccgcctcccc gcat
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
名称：pHR_Gal4UAS_Pembrolizumab_heavychain_T2A_ Pembrolizumab_lightchain_PGK_mCherry哺乳抗体质粒
别称: Plasmid#85434
| 启动子: | Gal4 |
|---|---|
| 复制子: | pUC |
| 原核抗性: | Amp |
| 筛选标记: | 红色荧光蛋白mCherry |
| 克隆菌株: | Stbl3 |
| 培养条件: | 37度 |
质粒属性
| 载体宿主： | 哺乳细胞,慢病毒 |
|---|---|
| 载体用途： | 蛋白表达,抗体表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 真核抗性： | |
| 荧光蛋白： | 红色 |
质粒简介
pHR_Gal4UAS_Pembrolizumab_heavychain_T2A_ Pembrolizumab_lightchain_PGK_mCherry是一个哺乳细胞抗体表达质粒。
质粒图谱
质粒序列
LOCUS Exported 12264 bp ds-DNA circular SYN 03-JAN-2016
FEATURES Location/Qualifiers
source 1..12264
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 96..7517
/label=match to pHR-CMVLacZ
/note="match to pHR-CMVLacZ"
/note="color: #a6acb3"
primer_bind complement(320..337)
/label=SP6
/note="SP6"
/note="color: #00ff00; direction: LEFT"
CDS 1231..2091
/codon_start=1
/label=bla (AmpR)
/note="bla"
/note="color: #993366"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
CDS 1429..2088
/label=AmpR
/note="AmpR"
/note="color: #ffff00; direction: RIGHT"
rep_origin 2186..2868
/label=ColE1 origin
/note="ColE1 origin"
/note="color: #808080"
promoter 3084..3410
/label=SV40 - Invitrogen
/note="SV40 - Invitrogen promoter"
/note="color: #0000ff"
promoter 3229..3425
/label=SV40 early promoter
/note="SV40 early promoter"
/note="color: #6495ed"
polyA_site 5105..5123
/label=SV40 pA
/note="SV40 pA"
/note="color: #ccff66"
polyA_signal complement(5138..5329)
/label=SV40 late polyA
/note="SV40 late polyA"
/note="color: #ffc0cb; direction: LEFT"
polyA_site 5199..5330
/label=SV40 early poly A
/note="SV40 early poly A"
/note="color: #80ff00"
polyA_site 5199..5329
/label=SV40 early poly A (2)
/note="SV40 early poly A"
/note="color: #66ff66"
repeat_region 7518..7697
/label=cPPT
/note="cPPT"
/note="color: #a6acb3"
primer_bind 7577..7598
/note="QB2741"
/note="color: #ff0000; direction: RIGHT"
primer_bind 7673..7696
/note="cPPT_5'seq"
/note="color: #a6acb3; direction: RIGHT"
misc_feature 7716..7721
/note="EcoRI"
/note="color: #a6acb3"
misc_feature 7722..7807
/note="Gal4"
/note="color: #a6acb3"
misc_feature 7828..7847
/note="Gal4"
/note="color: #a6acb3"
misc_feature 7848..7853
/note="SpeI"
/note="color: #a6acb3"
promoter 7854..7971
/note="mCMV"
/note="color: #ffffff"
misc_feature 7972..7991
/note="20BPhomo BamHI-pHR Gal4UAS synNotch_fwd"
/note="color: #a6acb3"
misc_feature 7992..9445
/note="Pembrolizumab_HC_gBlock"
/note="color: #a6acb3"
misc_feature 7992..7997
/note="Kozak Sequence"
/note="color: #ff00ff"
misc_feature 7998..8054
/note="Human IgG Heavy Chain Signal Peptide"
/note="Kober et al 2013 Biotech and Bioeng
Codon Optimized"
/note="color: #ff0000"
misc_feature 8055..8084
/note="myc tag"
/note="label=myc tag - ApEinfo_fwdcolor=#ffff80 -
ApEinfo_revcolor=green - ApEinfo_graphicformat=arrow_data
{{0 1 2 0 0 -1} {} 0} - width 5 offset 0 - 165..194"
/note="color: #dc143c; direction: RIGHT"
misc_feature 8055..8084
/note="cw35"
/note="label=cw35 - ApEinfo_fwdcolor=#8080c0 -
ApEinfo_revcolor=green - ApEinfo_graphicformat=arrow_data
{{0 1 2 0 0 -1} {} 0} - width 5 offset 0 - 165..214"
/note="color: #dc143c; direction: RIGHT"
misc_feature complement(8055..8084)
/note="cw34"
/note="label=cw34 - ApEinfo_fwdcolor=cyan -
ApEinfo_revcolor=green - ApEinfo_graphicformat=arrow_data
{{0 1 2 0 0 -1} {} 0} - width 5 offset 0 -
complement(145..194)"
/note="color: #00ffff; direction: LEFT"
misc_feature 8055..8084
/product="Myc (human c-Myc oncogene) epitope tag"
/note="Myc"
/note="color: #cc99b2"
misc_feature 8085..9425
/note="Pembrolizumab Heavy Chain"
/note="IDT Codon Optimized (gBlocks setting, homo sapiens)
From AA :
http://www.drugbank.ca/drugs/DB09037/polypeptide_sequences.
fasta"
/note="color: #00ff00"
misc_feature 8085..8444
/note="Pembrolizumab HC Variable"
/note="color: #0000ff"
misc_feature 8445..9425
/note="huIgG4 Constant"
/note=" (http://www.uniprot.org/uniprot/P01861)"
/note="color: #00ffff"
misc_feature 8766..8768
/note="S -> P"
/note="color: #993366"
misc_feature 9426..10244
/note="Pembrolizumab_LC_gBlock"
/note="color: #a6acb3"
misc_feature 9426..9560
/note="mAb_Center_gBlock"
/note="color: #ffcc99"
misc_feature 9426..9437
/note="Furin Cleavage Site"
/note="color: #ff0000"
misc_feature 9438..9449
/note="SGSG Spacer"
/note="color: #a6acb3"
misc_feature 9450..9503
/note="T2A"
/note="Codon optimized from AA sequence from Yang et al.
2008"
/note="color: #ffcc00"
misc_feature 9504..9560
/note="Human IgG Light Chain Signal Peptide"
/note="Kober et al 2013 Biotech and Bioeng
Codon Optimized"
/note="color: #ff0000"
misc_feature 9561..9587
/note="HA Tag"
/note="Sequence from Wiki"
/note="color: #ff99cc"
misc_feature 9588..10241
/note="Pembrolizumab Light Chain"
/note="IDT Codon Optimized (gBlock setting, homo sapien)
From AA :
http://www.drugbank.ca/drugs/DB09037/polypeptide_sequences.
fasta"
/note="color: #99cc00"
misc_feature 9588..9923
/note="Pembrolizumab LC Variable"
/note="color: #ff99cc"
misc_feature 9924..10241
/note="Ig kappa chain C region"
/note="color: #ccffcc"
misc_feature 10367..10866
/note="promPGK"
/note="color: #996633; direction: RIGHT"
misc_feature 10887..11597
/note="mCherry"
/note="color: #ff0000; direction: RIGHT"
enhancer 11673..12264
/label=WPRE
/note="WPRE"
/note="color: #a6acb3"
ORIGIN
1 cgataccgtc gaccaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
61 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
121 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
181 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
241 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatct
301 agaattaatt ccgtgtattc tatagtgtca cctaaatcgt atgtgtatga tacataaggt
361 tatgtattaa ttgtagccgc gttctaacga caatatgtac aagcctaatt gtgtagcatc
421 tggcttactg aagcagaccc tatcatctct ctcgtaaact gccgtcagag tcggtttggt
481 tggacgaacc ttctgagttt ctggtaacgc cgtcccgcac ccggaaatgg tcagcgaacc
541 aatcagcagg gtcatcgcta gccagatcct ctacgccgga cgcatcgtgg ccggcatcac
601 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
661 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
721 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
781 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
841 tcgaatggtg cactctcagt acaatctagc tctgatgccg catagttaag ccagccccga
901 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
961 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
1021 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
1081 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
1141 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
1201 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
1261 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
1321 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
1381 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
1441 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
1501 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
1561 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
1621 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
1681 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
1741 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
1801 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
1861 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
1921 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
1981 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
2041 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
2101 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
2161 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
2221 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
2281 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
2341 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
2401 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
2461 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
2521 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
2581 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
2641 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
2701 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
2761 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
2821 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
2881 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
2941 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
3001 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
3061 gcgcgttggc cgattcatta atgcagctgt ggaatgtgtg tcagttaggg tgtggaaagt
3121 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
3181 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
3241 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
3301 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
3361 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
3421 gcaaaaagct tggacacaag acaggcttgc gagatatgtt tgagaatacc actttatccc
3481 gcgtcaggga gaggcagtgc gtaaaaagac gcggactcat gtgaaatact ggtttttagt
3541 gcgccagatc tctataatct cgcgcaacct attttcccct cgaacacttt ttaagccgta
3601 gataaacagg ctgggacact tcacatgagc gaaaaataca tcgtcacctg ggacatgttg
3661 cagatccatg cacgtaaact cgcaagccga ctgatgcctt ctgaacaatg gaaaggcatt
3721 attgccgtaa gccgtggcgg tctgtaccgg gtgcgttact ggcgcgtgaa ctgggtattc
3781 gtcatgtcga taccgtttgt atttccagct acgatcacga caaccagcgc gagcttaaag
3841 tgctgaaacg cgcagaaggc gatggcgaag gcttcatcgt tattgatgac ctggtggata
3901 ccggtggtac tgcggttgcg attcgtgaaa tgtatccaaa agcgcacttt gtcaccatct
3961 tcgcaaaacc ggctggtcgt ccgctggttg atgactatgt tgttgatatc ccgcaagata
4021 cctggattga acagccgtgg gatatgggcg tcgtattcgt cccgccaatc tccggtcgct
4081 aatcttttca acgcctggca ctgccgggcg ttgttctttt taacttcagg cgggttacaa
4141 tagtttccag taagtattct ggaggctgca tccatgacac aggcaaacct gagcgaaacc
4201 ctgttcaaac cccgctttaa acatcctgaa acctcgacgc tagtccgccg ctttaatcac
4261 ggcgcacaac cgcctgtgca gtcggccctt gatggtaaaa ccatccctca ctggtatcgc
4321 atgattaacc gtctgatgtg gatctggcgc ggcattgacc cacgcgaaat cctcgacgtc
4381 caggcacgta ttgtgatgag cgatgccgaa cgtaccgacg atgatttata cgatacggtg
4441 attggctacc gtggcggcaa ctggatttat gagtgggccc cggatctttg tgaaggaacc
4501 ttacttctgt ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt
4561 aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt
4621 ttagattcca acctatggaa ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga
4681 aaacctgttt tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgactctca
4741 acattctact cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga
4801 attgctaagt tttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat
4861 ttacaccaca aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatattctgt
4921 aacctttata agtaggcata acagttataa tcataacata ctgttttttc ttactccaca
4981 caggcataga gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt
5041 aatttgtaaa ggggttaata aggaatattt gatgtatagt gccttgacta gagatcataa
5101 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
5161 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
5221 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
5281 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga
5341 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca
5401 attacctagt ggtttcattt actctaaacc tgtgattcct ctgaattatt ttcattttaa
5461 agaaattgta tttgttaaat atgtactaca aacttagtag ttggaagggc taattcactc
5521 ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
5581 ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta
5641 caagctagta ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag
5701 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg
5761 gaggtttgac agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt
5821 caagaactgc tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag
5881 gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc
5941 tttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
6001 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
6061 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
6121 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
6181 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
6241 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
6301 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
6361 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
6421 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
6481 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
6541 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
6601 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg gtgatcttca
6661 gacctggacg atatatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
6721 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
6781 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
6841 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
6901 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
6961 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
7021 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
7081 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
7141 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
7201 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
7261 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
7321 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
7381 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
7441 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
7501 gaacggatct cgacggtcgc caaatggcag tattcatcca caattttaaa agaaaagggg
7561 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
7621 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
7681 gcagagatcc agtttggatc gataagcttg atatcgaatt cggagcactg tcctccgaac
7741 gtcggagcac tgtcctccga acgtcggagc actgtcctcc gaacgtcgga gcactgtcct
7801 ccgaacggag catgtcctcc gaacgtcgga gcactgtcct ccgaacgact agttaggcgt
7861 gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
7921 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctctcgaca
7981 ttcgttggat cgccgccatg gattggacat ggagagtgtt ttgtctgctt gcggtgacac
8041 ctggcgctca tcctgagcag aagctgatca gcgaggagga cctgcaggta caactggtcc
8101 agtcaggcgt cgaggttaaa aagcccggtg cctcagtgaa ggtctcctgc aaagccagtg
8161 gttacacctt cactaattac tatatgtatt gggtcagaca ggcacccggc cagggtcttg
8221 aatggatggg aggcatcaat cccagcaatg gagggacaaa cttcaatgaa aagttcaaaa
8281 atcgcgtgac cctcacaacc gactcctcca caacaacagc atatatggaa ctgaagtctc
8341 ttcagtttga tgacaccgcc gtctactact gcgcccgacg agactaccgg ttcgatatgg
8401 gattcgacta ttggggtcag gggacgacag tgacagtctc ctccgcatct acaaagggac
8461 catcagtgtt tcctctggcc ccgtgctccc gctctaccag cgaatctacc gcagcattgg
8521 gctgtttggt gaaggactac ttccctgagc cagtgaccgt gtcctggaac agcggggcac
8581 tcactagcgg agtgcacaca tttccagccg tgcttcaaag cagcggtctc tattccctct
8641 ccagcgtggt gactgtgcca tccagctctc tcggcacgaa gacctatacc tgcaacgtgg
8701 accacaagcc ttcaaatacc aaggtggata aacgcgtcga atccaaatat ggcccaccct
8761 gcccgccatg tccagctcca gagtttctcg gcgggcctag cgtgttcttg tttcccccca
8821 aacccaagga cacgctcatg atttccagaa cacctgaagt tacctgcgtc gtcgtagacg
8881 tcagtcagga ggacccggaa gttcagttta actggtacgt ggacggtgtt gaggtgcata
8941 atgccaaaac caagccaaga gaagagcagt tcaattctac atatcgagtg gtgtcagtcc
9001 tgacagttct ccaccaggac tggttgaacg gcaaagagta caaatgcaag gtctccaaca
9061 aaggattgcc gagcagtata gagaaaacaa ttagtaaagc taagggtcag cccagagagc
9121 cccaggtgta cacgttgcca ccttcacagg aagagatgac gaagaaccaa gtgtctctga
9181 cctgcctggt gaagggcttc tacccatcag acattgcagt ggagtgggaa agcaatgggc
9241 agcctgagaa caactacaaa actacacccc ccgtcctgga cagcgatggc agtttcttcc
9301 tgtactcaag gttgactgtg gacaaaagca gatggcagga aggcaatgtg ttcagctgta
9361 gtgtgatgca cgaggccctc cacaatcatt acactcagaa atccctctct ctgagcctgg
9421 gaaagaggaa gcgacggagt gggtcaggcg aggggagagg ctcactcctc acgtgtggag
9481 acgttgaaga aaaccccgga ccaatggctt ggagtcctct gttcctgacc ctgattactc
9541 actgcgctgg aagctgggct tacccatacg atgttccaga ttacgctgag attgttctga
9601 cccaatctcc tgcaacactg agcctgtccc ccggagagag ggcaaccctc tcctgccgcg
9661 ccagtaaggg agtgtctacg tctggatatt cctatctcca ttggtatcag cagaagcctg
9721 gccaggcgcc caggcttctc atctaccttg cttcctatct ggagtccggg gtgccagccc
9781 gcttttctgg gtccggctct ggtaccgact tcaccctgac aatttcctca ctggagcctg
9841 aggatttcgc tgtctactat tgtcaacatt ccagagacct tcccctgacc tttggagggg
9901 gtacaaaagt ggagataaaa cgaacagtgg ccgccccatc tgtattcatc tttcctccat
9961 ctgatgaaca gcttaagtcc ggcactgctt ctgttgtttg cctgcttaac aatttttacc
10021 cgcgggaagc caaggtgcaa tggaaagtgg acaacgccct gcagtctggt aacagtcagg
10081 aaagcgtgac tgaacaggac agcaaagact ccacatactc cttgagttcc accttgacgc
10141 tcagtaaggc agactacgag aagcacaagg tttatgcctg tgaagtgact caccagggcc
10201 tctctagccc agtgactaag tccttcaacc gcggggagtg ttgagatcct tgacttgcgg
10261 ccgcaactcc cacctgcaac atgcgtgact gactgaggcc gcgactctag agtcgacctg
10321 catctaggcg ccggaattag atctctcgag gttaacgaat tctaccgggt aggggaggcg
10381 cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac
10441 acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg
10501 ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc
10561 gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc
10621 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa
10681 tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc
10741 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
10801 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc
10861 ctttcgacct gcagcccaag cttaccatgg tgagcaaggg cgaggaggat aacatggcca
10921 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
10981 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
11041 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
11101 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
11161 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
11221 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
11281 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
11341 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
11401 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
11461 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg
11521 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
11581 acgagctgta caagtaaagc ggccgcgact ctagagtcga cctgcaggca tgcaagcttg
11641 caggcatgca agcttgatat caagcttatc gataatcaac ctctggatta caaaatttgt
11701 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
11761 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
11821 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
11881 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
11941 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
12001 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
12061 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
12121 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
12181 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
12241 tccctttggg ccgcctcccc gcat
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
| P0540/pENTR223-FMR1人源基因质粒 |
| P0620/pENTR223-MS4A1人源基因质粒 |
| P1844/pENTR223-MAPK15人源基因质粒 |
| P1845/pENTR223-HMGN3人源基因质粒 |
| P1860/pENTR223-IMAGE3462332人源基因质粒 |
| P1880/pENTR223-ICMT人源基因质粒 |
| P1895/pENTR223-PIP4K2B人源基因质粒 |
| P1896/pENTR223-LOC142937人源基因质粒 |
| P1897/pENTR223-INIP人源基因质粒 |
| P1898/pENTR223-SPCS1人源基因质粒 |
| P1899/pENTR223-ANAPC16人源基因质粒 |
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pHR_Gal4UAS_Pembrolizumab_heavychain_T2A_ Pembrolizumab_lightchain_PGK_mCherry哺乳抗体质粒
￥100 - 1000
