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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#105815
- 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：pC0056 - LwCas13a-msfGFP-NES哺乳编辑质粒
别称: Plasmid#105815
质粒属性
质粒简介
pC0056 - LwCas13a-msfGFP-NES哺乳编辑质粒
质粒图谱
质粒序列
LOCUS Exported 13585 bp ds-DNA circular SYN 08-JUL-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pC0056 - LwCas13a-msfGFP-NES
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13585)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..13585
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/note="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
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2682..3860
/note="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
misc_feature 3876..3908
/note="NES"
CDS 3915..7370
/codon_start=1
/product="Leptotrichia wadei CRISPR-Cas effector that acts
as an RNA-guided RNAse (Abudayyeh et al., 2017)"
/note="LwCas13a"
/note="previously called C2c2"
/translation="MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIY
IKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEY
DLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINEN
NVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEK
YKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLD
KEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLL
NKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETE
NENGITGRMRGKTVKNNKGEEKYVSGEVDKIYN-ENKQNEVKENLKMFYSYDFNMDNKNE
IEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLK
IFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFW
SVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQ
KFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIE
SNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKY
TENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDF
ELEANEIGKFLDFN-ENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEK
IADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAI
GNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE
IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNY
IAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKK
IEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE"
misc_feature 7371..7415
/note="linker"
misc_feature 7416..8129
/note="sfGFP"
misc_feature 8136..8168
/note="NES"
CDS 8169..8249
/codon_start=1
/product="three tandem HA epitope tags"
/note="3xHA"
/translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
misc_feature 8277..8865
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 9390..9570
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 9602..9826
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 9872..10300
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10314..10643
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 10494..10629
/note="SV40 ori"
/note="SV40 origin of replication"
promoter 10691..10738
/note="EM7 promoter"
/note="synthetic bacterial promoter "
CDS 10757..11131
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/note="Zeo"
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 11261..11382
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(11833..12421)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12592..13452)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(13453..13557)
/gene="bla"
/note="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 atgcaaagat ggataaagtt ttaaacagag aggaatcttt
2641 gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
2701 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
2761 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
2821 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
2881 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
2941 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
3001 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
3061 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
3121 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
3181 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
3241 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
3301 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
3361 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
3421 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
3481 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
3541 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
3601 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
3661 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
3721 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
3781 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
3841 ttccatttca ggtgtcgtga cgtacggcca ccatgctgta ccctgagcgg ctgcggcgga
3901 tcctgaccgg atccatgaaa gtgaccaagg tcgacggcat cagccacaag aagtacatcg
3961 aagagggcaa gctcgtgaag tccaccagcg aggaaaaccg gaccagcgag agactgagcg
4021 agctgctgag catccggctg gacatctaca tcaagaaccc cgacaacgcc tccgaggaag
4081 agaaccggat cagaagagag aacctgaaga agttctttag caacaaggtg ctgcacctga
4141 aggacagcgt gctgtatctg aagaaccgga aagaaaagaa cgccgtgcag gacaagaact
4201 atagcgaaga ggacatcagc gagtacgacc tgaaaaacaa gaacagcttc tccgtgctga
4261 agaagatcct gctgaacgag gacgtgaact ctgaggaact ggaaatcttt cggaaggacg
4321 tggaagccaa gctgaacaag atcaacagcc tgaagtacag cttcgaagag aacaaggcca
4381 actaccagaa gatcaacgag aacaacgtgg aaaaagtggg cggcaagagc aagcggaaca
4441 tcatctacga ctactacaga gagagcgcca agcgcaacga ctacatcaac aacgtgcagg
4501 aagccttcga caagctgtat aagaaagagg atatcgagaa actgtttttc ctgatcgaga
4561 acagcaagaa gcacgagaag tacaagatcc gcgagtacta tcacaagatc atcggccgga
4621 agaacgacaa agagaacttc gccaagatta tctacgaaga gatccagaac gtgaacaaca
4681 tcaaagagct gattgagaag atccccgaca tgtctgagct gaagaaaagc caggtgttct
4741 acaagtacta cctggacaaa gaggaactga acgacaagaa tattaagtac gccttctgcc
4801 acttcgtgga aatcgagatg tcccagctgc tgaaaaacta cgtgtacaag cggctgagca
4861 acatcagcaa cgataagatc aagcggatct tcgagtacca gaatctgaaa aagctgatcg
4921 aaaacaaact gctgaacaag ctggacacct acgtgcggaa ctgcggcaag tacaactact
4981 atctgcaagt gggcgagatc gccacctccg actttatcgc ccggaaccgg cagaacgagg
5041 ccttcctgag aaacatcatc ggcgtgtcca gcgtggccta cttcagcctg aggaacatcc
5101 tggaaaccga gaacgagaac ggtatcaccg gccggatgcg gggcaagacc gtgaagaaca
5161 acaagggcga agagaaatac gtgtccggcg aggtggacaa gatctacaat gagaacaagc
5221 agaacgaagt gaaagaaaat ctgaagatgt tctacagcta cgacttcaac atggacaaca
5281 agaacgagat cgaggacttc ttcgccaaca tcgacgaggc catcagcagc atcagacacg
5341 gcatcgtgca cttcaacctg gaactggaag gcaaggacat cttcgccttc aagaatatcg
5401 cccccagcga gatctccaag aagatgtttc agaacgaaat caacgaaaag aagctgaagc
5461 tgaaaatctt caagcagctg aacagcgcca acgtgttcaa ctactacgag aaggatgtga
5521 tcatcaagta cctgaagaat accaagttca acttcgtgaa caaaaacatc cccttcgtgc
5581 ccagcttcac caagctgtac aacaagattg aggacctgcg gaataccctg aagttttttt
5641 ggagcgtgcc caaggacaaa gaagagaagg acgcccagat ctacctgctg aagaatatct
5701 actacggcga gttcctgaac aagttcgtga aaaactccaa ggtgttcttt aagatcacca
5761 atgaagtgat caagattaac aagcagcgga accagaaaac cggccactac aagtatcaga
5821 agttcgagaa catcgagaaa accgtgcccg tggaatacct ggccatcatc cagagcagag
5881 agatgatcaa caaccaggac aaagaggaaa agaataccta catcgacttt attcagcaga
5941 ttttcctgaa gggcttcatc gactacctga acaagaacaa tctgaagtat atcgagagca
6001 acaacaacaa tgacaacaac gacatcttct ccaagatcaa gatcaaaaag gataacaaag
6061 agaagtacga caagatcctg aagaactatg agaagcacaa tcggaacaaa gaaatccctc
6121 acgagatcaa tgagttcgtg cgcgagatca agctggggaa gattctgaag tacaccgaga
6181 atctgaacat gttttacctg atcctgaagc tgctgaacca caaagagctg accaacctga
6241 agggcagcct ggaaaagtac cagtccgcca acaaagaaga aaccttcagc gacgagttgg
6301 aactgatcaa cctgctgaac ctggacaaca acagagtgac cgaggacttc gagctggaag
6361 ccaacgagat cggcaagttc ctggacttca acgaaaacaa aatcaaggac cggaaagagc
6421 tgaaaaagtt cgacaccaac aagatctatt tcgacggcga gaacatcatc aagcaccggg
6481 ccttctacaa tatcaagaaa tacggcatgc tgaatctgct ggaaaagatc gccgataagg
6541 ccaagtataa gatcagcctg aaagaactga aagagtacag caacaagaag aatgagattg
6601 aaaagaacta caccatgcag cagaacctgc accggaagta cgccagaccc aagaaggacg
6661 aaaagttcaa cgacgaggac tacaaagagt atgagaaggc catcggcaac atccagaagt
6721 acacccacct gaagaacaag gtggaattca atgagctgaa cctgctgcag ggcctgctgc
6781 tgaagatcct gcaccggctc gtgggctaca ccagcatctg ggagcgggac ctgagattcc
6841 ggctgaaggg cgagtttccc gagaaccact acatcgagga aattttcaat ttcgacaact
6901 ccaagaatgt gaagtacaaa agcggccaga tcgtggaaaa gtatatcaac ttctacaaag
6961 aactgtacaa ggacaatgtg gaaaagcgga gcatctactc cgacaagaaa gtgaagaaac
7021 tgaagcagga aaaaaaggac ctgtacatcc ggaactacat tgcccacttc aactacatcc
7081 cccacgccga gattagcctg ctggaagtgc tggaaaacct gcggaagctg ctgtcctacg
7141 accggaagct gaagaacgcc atcatgaagt ccatcgtgga cattctgaaa gaatacggct
7201 tcgtggccac cttcaagatc ggcgctgaca agaagatcga aatccagacc ctggaatcag
7261 agaagatcgt gcacctgaag aatctgaaga aaaagaaact gatgaccgac cggaacagcg
7321 aggaactgtg cgaactcgtg aaagtcatgt tcgagtacaa ggccctggaa ggaggaggtg
7381 gaagcggagg aggaggaagc ggaggaggag gtagcgtgag taaaggtgaa gaactcttca
7441 ctggagtagt gcccattctg gtagagcttg atggagatgt aaatggacat aaattctccg
7501 tcaggggcga aggcgaaggg gacgccacga atggtaagct gactctgaaa ttcatctgta
7561 cgacgggcaa actgcccgtc ccatggccta cactcgtaac gaccctcacc tacggcgtgc
7621 aatgcttttc tcgatatccc gaccacatga aacagcatga ctttttcaag tctgcaatgc
7681 ctgaaggtta tgttcaagaa aggaccatca gctttaagga tgatggtaca tataaaaccc
7741 gagccgaggt taaatttgaa ggggacactc tggttaatcg aattgaactg aaaggtattg
7801 attttaagga ggacggtaac atactggggc acaagttgga gtacaacttt aacagccata
7861 atgtgtatat taccgctgat aagcagaaaa atgggataaa ggccaacttt aagatccgac
7921 ataatgtcga agatggtagt gttcaactgg ctgatcatta ccaacaaaat acgcccatcg
7981 gagatggacc tgtactcttg cctgacaatc attatctctc cacgcaatca aagctttcca
8041 aggacccaaa cgaaaagaga gatcacatgg tccttctgga atttgtgact gccgcaggca
8101 tcactctcgg tatggatgag ctgtacaagg gatccctgta tcctgagcgg ctgcggcgga
8161 tcctgaccta cccatacgat gttccagatt acgcttatcc ctacgacgtg cctgattatg
8221 catacccata tgatgtcccc gactatgcct aagaattcga tatcaagctt atcgataatc
8281 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
8341 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
8401 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
8461 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
8521 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
8581 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
8641 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
8701 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
8761 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
8821 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac
8881 ctcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg
8941 attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg
9001 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
9061 aggggggact ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga
9121 tctaccacac acaaggctac ttccctgatt ggcagaacta cacaccaggg ccagggatca
9181 gatatccact gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag
9241 aagaagccaa tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg
9301 atgacccgga gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca
9361 tggcccgaga gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct
9421 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
9481 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
9541 ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg
9601 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
9661 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
9721 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
9781 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
9841 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
9901 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
9961 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
10021 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
10081 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
10141 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
10201 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
10261 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
10321 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
10381 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
10441 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
10501 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
10561 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
10621 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
10681 atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
10741 ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
10801 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
10861 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
10921 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
10981 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
11041 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
11101 cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
11161 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
11221 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
11281 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
11341 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
11401 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
11461 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
11521 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
11581 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
11641 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
11701 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
11761 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
11821 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
11881 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
11941 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
12001 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
12061 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
12121 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
12181 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
12241 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
12301 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
12361 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
12421 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
12481 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
12541 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
12601 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
12661 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
12721 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
12781 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
12841 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
12901 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
12961 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
13021 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
13081 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
13141 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
13201 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
13261 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
13321 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
13381 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
13441 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
13501 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
13561 atttccccga aaagtgccac ctgac
//
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：pC0056 - LwCas13a-msfGFP-NES哺乳编辑质粒
别称: Plasmid#105815
| 复制子: | pUC |
|---|---|
| 原核抗性: | Amp |
| 克隆菌株: | DH5a |
| 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 |
| 原核抗性： | Amp |
| 真核抗性： | |
| 荧光蛋白： |
质粒简介
pC0056 - LwCas13a-msfGFP-NES哺乳编辑质粒
质粒图谱
质粒序列
LOCUS Exported 13585 bp ds-DNA circular SYN 08-JUL-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pC0056 - LwCas13a-msfGFP-NES
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13585)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..13585
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/note="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
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2682..3860
/note="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
misc_feature 3876..3908
/note="NES"
CDS 3915..7370
/codon_start=1
/product="Leptotrichia wadei CRISPR-Cas effector that acts
as an RNA-guided RNAse (Abudayyeh et al., 2017)"
/note="LwCas13a"
/note="previously called C2c2"
/translation="MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIY
IKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEY
DLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINEN
NVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEK
YKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLD
KEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLL
NKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETE
NENGITGRMRGKTVKNNKGEEKYVSGEVDKIYN-ENKQNEVKENLKMFYSYDFNMDNKNE
IEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLK
IFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFW
SVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQ
KFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIE
SNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKY
TENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDF
ELEANEIGKFLDFN-ENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEK
IADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAI
GNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE
IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNY
IAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKK
IEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE"
misc_feature 7371..7415
/note="linker"
misc_feature 7416..8129
/note="sfGFP"
misc_feature 8136..8168
/note="NES"
CDS 8169..8249
/codon_start=1
/product="three tandem HA epitope tags"
/note="3xHA"
/translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
misc_feature 8277..8865
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 9390..9570
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 9602..9826
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 9872..10300
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10314..10643
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 10494..10629
/note="SV40 ori"
/note="SV40 origin of replication"
promoter 10691..10738
/note="EM7 promoter"
/note="synthetic bacterial promoter "
CDS 10757..11131
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/note="Zeo"
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 11261..11382
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(11833..12421)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12592..13452)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(13453..13557)
/gene="bla"
/note="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 atgcaaagat ggataaagtt ttaaacagag aggaatcttt
2641 gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
2701 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
2761 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
2821 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
2881 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
2941 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
3001 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
3061 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
3121 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
3181 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
3241 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
3301 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
3361 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
3421 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
3481 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
3541 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
3601 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
3661 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
3721 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
3781 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
3841 ttccatttca ggtgtcgtga cgtacggcca ccatgctgta ccctgagcgg ctgcggcgga
3901 tcctgaccgg atccatgaaa gtgaccaagg tcgacggcat cagccacaag aagtacatcg
3961 aagagggcaa gctcgtgaag tccaccagcg aggaaaaccg gaccagcgag agactgagcg
4021 agctgctgag catccggctg gacatctaca tcaagaaccc cgacaacgcc tccgaggaag
4081 agaaccggat cagaagagag aacctgaaga agttctttag caacaaggtg ctgcacctga
4141 aggacagcgt gctgtatctg aagaaccgga aagaaaagaa cgccgtgcag gacaagaact
4201 atagcgaaga ggacatcagc gagtacgacc tgaaaaacaa gaacagcttc tccgtgctga
4261 agaagatcct gctgaacgag gacgtgaact ctgaggaact ggaaatcttt cggaaggacg
4321 tggaagccaa gctgaacaag atcaacagcc tgaagtacag cttcgaagag aacaaggcca
4381 actaccagaa gatcaacgag aacaacgtgg aaaaagtggg cggcaagagc aagcggaaca
4441 tcatctacga ctactacaga gagagcgcca agcgcaacga ctacatcaac aacgtgcagg
4501 aagccttcga caagctgtat aagaaagagg atatcgagaa actgtttttc ctgatcgaga
4561 acagcaagaa gcacgagaag tacaagatcc gcgagtacta tcacaagatc atcggccgga
4621 agaacgacaa agagaacttc gccaagatta tctacgaaga gatccagaac gtgaacaaca
4681 tcaaagagct gattgagaag atccccgaca tgtctgagct gaagaaaagc caggtgttct
4741 acaagtacta cctggacaaa gaggaactga acgacaagaa tattaagtac gccttctgcc
4801 acttcgtgga aatcgagatg tcccagctgc tgaaaaacta cgtgtacaag cggctgagca
4861 acatcagcaa cgataagatc aagcggatct tcgagtacca gaatctgaaa aagctgatcg
4921 aaaacaaact gctgaacaag ctggacacct acgtgcggaa ctgcggcaag tacaactact
4981 atctgcaagt gggcgagatc gccacctccg actttatcgc ccggaaccgg cagaacgagg
5041 ccttcctgag aaacatcatc ggcgtgtcca gcgtggccta cttcagcctg aggaacatcc
5101 tggaaaccga gaacgagaac ggtatcaccg gccggatgcg gggcaagacc gtgaagaaca
5161 acaagggcga agagaaatac gtgtccggcg aggtggacaa gatctacaat gagaacaagc
5221 agaacgaagt gaaagaaaat ctgaagatgt tctacagcta cgacttcaac atggacaaca
5281 agaacgagat cgaggacttc ttcgccaaca tcgacgaggc catcagcagc atcagacacg
5341 gcatcgtgca cttcaacctg gaactggaag gcaaggacat cttcgccttc aagaatatcg
5401 cccccagcga gatctccaag aagatgtttc agaacgaaat caacgaaaag aagctgaagc
5461 tgaaaatctt caagcagctg aacagcgcca acgtgttcaa ctactacgag aaggatgtga
5521 tcatcaagta cctgaagaat accaagttca acttcgtgaa caaaaacatc cccttcgtgc
5581 ccagcttcac caagctgtac aacaagattg aggacctgcg gaataccctg aagttttttt
5641 ggagcgtgcc caaggacaaa gaagagaagg acgcccagat ctacctgctg aagaatatct
5701 actacggcga gttcctgaac aagttcgtga aaaactccaa ggtgttcttt aagatcacca
5761 atgaagtgat caagattaac aagcagcgga accagaaaac cggccactac aagtatcaga
5821 agttcgagaa catcgagaaa accgtgcccg tggaatacct ggccatcatc cagagcagag
5881 agatgatcaa caaccaggac aaagaggaaa agaataccta catcgacttt attcagcaga
5941 ttttcctgaa gggcttcatc gactacctga acaagaacaa tctgaagtat atcgagagca
6001 acaacaacaa tgacaacaac gacatcttct ccaagatcaa gatcaaaaag gataacaaag
6061 agaagtacga caagatcctg aagaactatg agaagcacaa tcggaacaaa gaaatccctc
6121 acgagatcaa tgagttcgtg cgcgagatca agctggggaa gattctgaag tacaccgaga
6181 atctgaacat gttttacctg atcctgaagc tgctgaacca caaagagctg accaacctga
6241 agggcagcct ggaaaagtac cagtccgcca acaaagaaga aaccttcagc gacgagttgg
6301 aactgatcaa cctgctgaac ctggacaaca acagagtgac cgaggacttc gagctggaag
6361 ccaacgagat cggcaagttc ctggacttca acgaaaacaa aatcaaggac cggaaagagc
6421 tgaaaaagtt cgacaccaac aagatctatt tcgacggcga gaacatcatc aagcaccggg
6481 ccttctacaa tatcaagaaa tacggcatgc tgaatctgct ggaaaagatc gccgataagg
6541 ccaagtataa gatcagcctg aaagaactga aagagtacag caacaagaag aatgagattg
6601 aaaagaacta caccatgcag cagaacctgc accggaagta cgccagaccc aagaaggacg
6661 aaaagttcaa cgacgaggac tacaaagagt atgagaaggc catcggcaac atccagaagt
6721 acacccacct gaagaacaag gtggaattca atgagctgaa cctgctgcag ggcctgctgc
6781 tgaagatcct gcaccggctc gtgggctaca ccagcatctg ggagcgggac ctgagattcc
6841 ggctgaaggg cgagtttccc gagaaccact acatcgagga aattttcaat ttcgacaact
6901 ccaagaatgt gaagtacaaa agcggccaga tcgtggaaaa gtatatcaac ttctacaaag
6961 aactgtacaa ggacaatgtg gaaaagcgga gcatctactc cgacaagaaa gtgaagaaac
7021 tgaagcagga aaaaaaggac ctgtacatcc ggaactacat tgcccacttc aactacatcc
7081 cccacgccga gattagcctg ctggaagtgc tggaaaacct gcggaagctg ctgtcctacg
7141 accggaagct gaagaacgcc atcatgaagt ccatcgtgga cattctgaaa gaatacggct
7201 tcgtggccac cttcaagatc ggcgctgaca agaagatcga aatccagacc ctggaatcag
7261 agaagatcgt gcacctgaag aatctgaaga aaaagaaact gatgaccgac cggaacagcg
7321 aggaactgtg cgaactcgtg aaagtcatgt tcgagtacaa ggccctggaa ggaggaggtg
7381 gaagcggagg aggaggaagc ggaggaggag gtagcgtgag taaaggtgaa gaactcttca
7441 ctggagtagt gcccattctg gtagagcttg atggagatgt aaatggacat aaattctccg
7501 tcaggggcga aggcgaaggg gacgccacga atggtaagct gactctgaaa ttcatctgta
7561 cgacgggcaa actgcccgtc ccatggccta cactcgtaac gaccctcacc tacggcgtgc
7621 aatgcttttc tcgatatccc gaccacatga aacagcatga ctttttcaag tctgcaatgc
7681 ctgaaggtta tgttcaagaa aggaccatca gctttaagga tgatggtaca tataaaaccc
7741 gagccgaggt taaatttgaa ggggacactc tggttaatcg aattgaactg aaaggtattg
7801 attttaagga ggacggtaac atactggggc acaagttgga gtacaacttt aacagccata
7861 atgtgtatat taccgctgat aagcagaaaa atgggataaa ggccaacttt aagatccgac
7921 ataatgtcga agatggtagt gttcaactgg ctgatcatta ccaacaaaat acgcccatcg
7981 gagatggacc tgtactcttg cctgacaatc attatctctc cacgcaatca aagctttcca
8041 aggacccaaa cgaaaagaga gatcacatgg tccttctgga atttgtgact gccgcaggca
8101 tcactctcgg tatggatgag ctgtacaagg gatccctgta tcctgagcgg ctgcggcgga
8161 tcctgaccta cccatacgat gttccagatt acgcttatcc ctacgacgtg cctgattatg
8221 catacccata tgatgtcccc gactatgcct aagaattcga tatcaagctt atcgataatc
8281 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
8341 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
8401 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
8461 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
8521 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
8581 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
8641 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
8701 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
8761 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
8821 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac
8881 ctcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg
8941 attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg
9001 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
9061 aggggggact ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga
9121 tctaccacac acaaggctac ttccctgatt ggcagaacta cacaccaggg ccagggatca
9181 gatatccact gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag
9241 aagaagccaa tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg
9301 atgacccgga gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca
9361 tggcccgaga gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct
9421 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
9481 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
9541 ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg
9601 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
9661 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
9721 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
9781 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
9841 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
9901 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
9961 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
10021 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
10081 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
10141 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
10201 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
10261 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
10321 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
10381 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
10441 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
10501 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
10561 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
10621 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
10681 atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
10741 ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
10801 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
10861 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
10921 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
10981 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
11041 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
11101 cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
11161 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
11221 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
11281 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
11341 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
11401 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
11461 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
11521 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
11581 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
11641 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
11701 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
11761 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
11821 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
11881 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
11941 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
12001 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
12061 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
12121 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
12181 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
12241 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
12301 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
12361 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
12421 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
12481 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
12541 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
12601 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
12661 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
12721 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
12781 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
12841 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
12901 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
12961 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
13021 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
13081 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
13141 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
13201 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
13261 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
13321 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
13381 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
13441 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
13501 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
13561 atttccccga aaagtgccac ctgac
//
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
| P2590/pENTR223-ASB6人源基因质粒 |
| P2591/pENTR223-ABHD13（409-1014bp）人源基因质粒 |
| P2592/pENTR223-TM4SF4人源基因质粒 |
| P2593/pENTR223-NMRK1人源基因质粒 |
| P2594/pENTR223-IL11人源基因质粒 |
| P2595/pENTR223-DLK2（538-1152bp）人源基因质粒 |
| P2596/pENTR223-ATP6V0B人源基因质粒 |
| P2597/pENTR223-RAB1A人源基因质粒 |
| P2598/pENTR223-MTHFS人源基因质粒 |
| P2599/pENTR223-ASF1B人源基因质粒 |
| P2600/pENTR223-DNAJC12-T19C人源基因质粒 |
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pC0056 - LwCas13a-msfGFP-NES哺乳编辑质粒
￥100 - 1000
