pCAG Cas9-2A-Citrine哺乳编辑质粒

基本信息
名称：pCAG Cas9-2A-Citrine哺乳编辑质粒
别称: Plasmid#92358

启动子:	CAG
复制子:	PUC
原核抗性:	Amp
克隆菌株:	Stbl3
培养条件:	37度

质粒属性

载体宿主：	哺乳细胞
载体用途：	基因敲除
基因种属：
基因类型：	CRISPR,Cas9
原核抗性：	Amp
真核抗性：
荧光蛋白：

质粒简介
 pCAG Cas9-2A-Citrine是一个哺乳细胞基因编辑Cas9表达质粒。

质粒图谱


质粒序列
LOCUS       Exported                9886 bp ds-DNA     circular SYN 06-JAN-2018
DEFINITION  synthetic circular DNA
FEATURES             Location/Qualifiers
     source          1..9886
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     regulatory      1789..1798
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1801..1821
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1846..5946
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNL-SDAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5947..5994
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6010..6063
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6070..6786
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_site      6943..6998
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     promoter        7562..7757
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7608..7743
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    7763..7897
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(8136..8724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8895..9755)
                     /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(9756..9860)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgct cgagagttcg
     1741 aagccttaag tacgcgtagg cgcgccttaa ttaacgttta aacgctaggc caccatggcc
     1801 ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc
     1861 atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
     1921 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
     1981 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga
     2041 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
     2101 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
     2161 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
     2221 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
     2281 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
     2341 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     2401 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
     2461 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
     2521 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg
     2581 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
     2641 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     2701 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
     2761 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
     2821 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
     2881 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga
     2941 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
     3001 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
     3061 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
     3121 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
     3181 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
     3241 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct
     3301 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag
     3361 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa
     3421 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag
     3481 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
     3541 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     3601 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     3661 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     3721 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     3781 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
     3841 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     3901 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
     3961 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
     4021 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
     4081 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
     4141 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
     4201 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
     4261 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
     4321 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt
     4381 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag
     4441 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg
     4501 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc
     4561 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
     4621 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat
     4681 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
     4741 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
     4801 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
     4861 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
     4921 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
     4981 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
     5041 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg
     5101 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
     5161 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg
     5221 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg
     5281 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
     5341 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
     5401 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
     5461 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg
     5521 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
     5581 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg
     5641 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
     5701 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc
     5761 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac
     5821 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc
     5881 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
     5941 ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaaggatatc
     6001 ggatcaggag agggcagggg aagtcttcta acatgcgggg acgtggagga aaatcccggg
     6061 cccagatcca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     6121 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     6181 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     6241 tggcccaccc tcgtgaccac cttcggctac ggcctgatgt gcttcgcccg ctaccccgac
     6301 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     6361 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     6421 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     6481 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     6541 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     6601 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     6661 gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     6721 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     6781 tacaagtaag gccgcaattc actcctcagg tgcaggctgc ctatcagaag gtggtggctg
     6841 gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt
     6901 atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt
     6961 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg
     7021 caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata
     7081 tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc
     7141 ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat
     7201 tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag
     7261 ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga
     7321 gatccctcga cctgcaccgt cgaccagctg gtcgacggtg caccgtcgac cagcttggcg
     7381 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     7441 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     7501 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc
     7561 cgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     7621 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     7681 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     7741 gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag
     7801 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     7861 gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa
     7921 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     7981 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     8041 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     8101 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     8161 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     8221 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     8281 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc
     8341 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     8401 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     8461 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     8521 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     8581 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     8641 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     8701 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     8761 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     8821 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     8881 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     8941 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     9001 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     9061 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     9121 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     9181 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     9241 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     9301 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     9361 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     9421 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     9481 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     9541 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     9601 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     9661 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     9721 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     9781 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     9841 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctg
//

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

P1321/pRSETB-mCherry大肠荧光质粒

P1582/pNCS-mCyRFP1大肠荧光质粒

P1675/pGEX-TagRFP大肠荧光质粒

P3013/pE2-Crimson大肠荧光质粒

P0027/pET28a-EYFP大肠荧光质粒

P1322/pRSETB-mVenus大肠荧光质粒

P1190/pT7-His-Venus大肠荧光质粒

P0542/pET22b-EBFP大肠荧光质粒

P1323/pRSETB-TagBFP大肠荧光质粒

P1324/pRSETB-mTurquoise2大肠荧光质粒

P0793/pRSET-ECFP大肠荧光质粒

P1071/mTurquoise2-pBAD大肠荧光质粒