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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
Lenti-Cas9-Puro
- 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：Lenti-Cas9-Puro哺乳编辑质粒
别称: Lenti-Cas9-Puro
质粒属性
质粒简介
Lenti-Cas9-Puro质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
LOCUS Exported 12700 bp ds-DNA circular SYN 27-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Lenti-Cas9-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12700)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 27, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..12700
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2440..2557
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2702..2913
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 2938..7041
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLS-DAILLS-DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 7042..7089
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 7090..7113
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 7123..7179
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 7180..7776
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 7792..8380
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 8452..8685
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 8717..8941
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 8987..9415
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 9429..9758
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 9609..9744
/label=SV40 ori
/note="SV40 origin of replication"
promoter 9806..9853
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 9872..10246
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=Zeo
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 10376..10497
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(10948..11536)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(11707..12567)
/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"
promoter complement(12568..12672)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
2581 agatccagtt tggttaatta aggtacccat atgcttaccg taacttgaaa gtatttcgat
2641 ttcttggaat tcgctagcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
2701 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
2761 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
2821 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
2881 ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc tgccaccatg
2941 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
3001 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
3061 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
3121 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
3181 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
3241 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
3301 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
3361 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
3421 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
3481 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
3541 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
3601 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
3661 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
3721 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
3781 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
3841 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
3901 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
3961 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
4021 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
4081 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
4141 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
4201 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
4261 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
4321 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
4381 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
4441 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
4501 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
4561 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
4621 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
4681 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
4741 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
4801 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
4861 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
4921 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
4981 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
5041 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
5101 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
5161 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
5221 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
5281 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
5341 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
5401 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
5461 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
5521 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
5581 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
5641 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
5701 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
5761 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
5821 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
5881 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
5941 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
6001 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
6061 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
6121 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
6181 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
6241 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
6301 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
6361 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
6421 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
6481 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
6541 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
6601 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
6661 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
6721 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
6781 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
6841 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
6901 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
6961 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
7021 ctgtctcagc tgggaggcga caagcgacct gccgccacaa agaaggctgg acaggctaag
7081 aagaagaaag attacaaaga cgatgacgat aagggatccg gcgcaacaaa cttctctctg
7141 ctgaaacaag ccggagatgt cgaagagaat cctggaccga ccgagtacaa gcccacggtg
7201 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
7261 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
7321 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
7381 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
7441 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
7501 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
7561 ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
7621 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
7681 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
7741 cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct
7801 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
7861 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
7921 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
7981 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
8041 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
8101 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
8161 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
8221 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
8281 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
8341 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
8401 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
8461 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
8521 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
8581 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
8641 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc
8701 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
8761 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
8821 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
8881 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
8941 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
9001 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
9061 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
9121 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
9181 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
9241 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
9301 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
9361 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
9421 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
9481 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
9541 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
9601 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
9661 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
9721 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
9781 ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat
9841 agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt
9901 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
9961 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
10021 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
10081 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
10141 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
10201 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga
10261 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
10321 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
10381 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
10441 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
10501 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
10561 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
10621 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
10681 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
10741 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
10801 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
10861 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
10921 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
10981 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
11041 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
11101 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
11161 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
11221 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
11281 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
11341 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
11401 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
11461 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
11521 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
11581 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
11641 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
11701 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
11761 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
11821 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
11881 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
11941 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
12001 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
12061 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
12121 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
12181 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
12241 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
12301 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
12361 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
12421 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
12481 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
12541 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
12601 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
12661 aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
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：Lenti-Cas9-Puro哺乳编辑质粒
别称: Lenti-Cas9-Puro
| 复制子: | pUC |
|---|---|
| 原核抗性: | Amp |
| 筛选标记: | Puro,Zeo |
| 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： | 基因敲除 |
| 基因种属： | |
| 基因类型： | |
| 原核抗性： | Amp |
| 真核抗性： | Puro,Zeo |
| 荧光蛋白： |
Lenti-Cas9-Puro质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
LOCUS Exported 12700 bp ds-DNA circular SYN 27-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Lenti-Cas9-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12700)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 27, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..12700
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2440..2557
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2702..2913
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 2938..7041
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLS-DAILLS-DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 7042..7089
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 7090..7113
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 7123..7179
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 7180..7776
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 7792..8380
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 8452..8685
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 8717..8941
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 8987..9415
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 9429..9758
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 9609..9744
/label=SV40 ori
/note="SV40 origin of replication"
promoter 9806..9853
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 9872..10246
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=Zeo
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 10376..10497
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(10948..11536)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(11707..12567)
/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"
promoter complement(12568..12672)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
2581 agatccagtt tggttaatta aggtacccat atgcttaccg taacttgaaa gtatttcgat
2641 ttcttggaat tcgctagcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
2701 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
2761 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
2821 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
2881 ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc tgccaccatg
2941 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
3001 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
3061 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
3121 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
3181 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
3241 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
3301 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
3361 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
3421 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
3481 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
3541 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
3601 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
3661 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
3721 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
3781 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
3841 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
3901 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
3961 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
4021 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
4081 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
4141 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
4201 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
4261 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
4321 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
4381 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
4441 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
4501 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
4561 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
4621 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
4681 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
4741 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
4801 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
4861 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
4921 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
4981 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
5041 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
5101 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
5161 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
5221 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
5281 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
5341 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
5401 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
5461 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
5521 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
5581 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
5641 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
5701 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
5761 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
5821 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
5881 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
5941 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
6001 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
6061 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
6121 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
6181 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
6241 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
6301 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
6361 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
6421 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
6481 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
6541 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
6601 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
6661 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
6721 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
6781 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
6841 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
6901 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
6961 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
7021 ctgtctcagc tgggaggcga caagcgacct gccgccacaa agaaggctgg acaggctaag
7081 aagaagaaag attacaaaga cgatgacgat aagggatccg gcgcaacaaa cttctctctg
7141 ctgaaacaag ccggagatgt cgaagagaat cctggaccga ccgagtacaa gcccacggtg
7201 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
7261 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
7321 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
7381 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
7441 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
7501 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
7561 ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
7621 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
7681 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
7741 cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct
7801 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
7861 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
7921 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
7981 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
8041 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
8101 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
8161 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
8221 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
8281 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
8341 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
8401 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
8461 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
8521 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
8581 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
8641 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc
8701 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
8761 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
8821 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
8881 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
8941 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
9001 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
9061 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
9121 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
9181 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
9241 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
9301 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
9361 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
9421 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
9481 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
9541 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
9601 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
9661 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
9721 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
9781 ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat
9841 agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt
9901 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
9961 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
10021 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
10081 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
10141 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
10201 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga
10261 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
10321 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
10381 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
10441 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
10501 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
10561 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
10621 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
10681 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
10741 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
10801 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
10861 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
10921 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
10981 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
11041 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
11101 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
11161 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
11221 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
11281 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
11341 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
11401 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
11461 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
11521 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
11581 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
11641 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
11701 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
11761 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
11821 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
11881 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
11941 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
12001 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
12061 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
12121 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
12181 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
12241 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
12301 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
12361 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
12421 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
12481 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
12541 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
12601 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
12661 aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
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
| P0076/pCold TF大肠表达质粒 |
| P0077/pCold-SUMO大肠表达质粒 |
| P0444/pCold-SUMO-10×His-3C-Strep II-FLAG大肠表达质粒 |
| P1100/pCold-ProS2大肠表达质粒 |
| P0337/pCold-GST大肠表达质粒 |
| P0694/pCold-MBP大肠表达质粒 |
| P0812/pBAD/Myc-HisA大肠表达质粒 |
| P0411/pBAD/Myc-HisB大肠表达质粒 |
| P0079/pBAD/His A大肠表达质粒 |
| P0080/pBAD/His B大肠表达质粒 |
| P0081/pBAD/His C大肠表达质粒 |
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CRISPR-Cas 系统是目前最常用的基因编辑工具，但是由于传统的 Cas 核酸酶分子量普遍太大，使其在在体基因治疗的应用中受限。近年来，为了解决这一难题，小的 Cas 核酸酶逐渐被发现和探究。其中，Cas12f 核酸酶是目前最紧凑的 CRISPR 效应核酸酶，比传统 Cas9 和 Cas12a 核酸酶小一半以上，在临床治疗应用中具有巨大潜力，然而高效的 Cas12f 基因编辑系统仍旧较少。 2022 年 9 月 27 日，上海科技大学季泉江教授团队在 Cell Reports 发表
诺奖得主 Science 发文：基因编辑诞生 10 年，未来将如何改变世界？
3D）。 目前，存在多种用于哺乳动物系统中 CRISPR 基因编辑的递送方式，广泛划分为进入物理递送、基于病毒的递送，以及基于合成材料的递送。常见的物理递送方法包括显微注射和电穿孔，允许控制剂量，效率递送高，但仅限于离体递送。基于病毒的递送方式包括腺病毒、腺相关病毒和慢病毒，它们以质粒 DNA 的形式通过载体传播，具有良好的效率。基于合成材料的递送方法更安全且有高水平的控制和灵活性，但其效率要低于病毒, 递送效率受到材料的体积和阳离子性质的限制，易导致间质分散不良。 因此，改进当前的递送
Lenti-Cas9-Puro哺乳编辑质粒
￥100 - 1000
