Product Information

Transformation of plasmid DNA to competent E. Coli cells

1.    Thaw competent cells on ice. 20–200µL per tube

2.    Add max. 1-3µL of plasmid

3.    Mix very gently!

4.    Incubate the tubes on ice for 30 min

5.    Heat shock the cells for 45 sec to 2 min at 42°C

6.    Place the tubes immediately on ice for at least 2 min

7.    Add 800µL of SOC medium to each tube

8.    Incubate for 1 hour at 37°C and shake vigorously

9.    Spin down briefly and remove most supernatants

10.  Resuspend cell pellet with the rest SOC medium in the tube by pipetting

11.  Plate out the suspension on a LB agar plate containing the appropriate antibiotic. Incubate the plates overnight at 37°C

Growth in Bacteria:

Bacterial Resistance(s)  Ampicillin

Growth Temperature  37°C

Growth Strain(s)  DH5alpha

Copy number  High Copy

Gene/Insert

Gene/Insert name  Cas9

Species  Streptococcus pyogenes

Insert Size (bp)  4500

GenBank ID  NC_002737.1

Promoter    CBh

LOCUS pU6-(BbsI)_CBh-Cas9-T2A-BFP-P2A-Ad4E1B   10810 bp DNA

DEFINITION .

ACCESSION

VERSION

SOURCE . (unknown)

 ORGANISM .

COMMENT

FEATURES Location/Qualifiers

 promoter 1..249

 /label=hU6

/label="hU6 promoter"

 Misc._feature 258..271

 /label=BbsI

 misc_feature 274..355

 /label="Guide RNA"

 /label="sgRNA scaffold"

 promoter 440..1238

 /label=CBh

/label="CBh promoter"

 CDS 1251..1319

 /label=3xFLAG

 CDS 1320..1370

 /label=NLS

 CDS 1371..5471

 /label=hSpCas9

 CDS 5472..5519

 /label=NLS(1)

 CDS 5529..5582

 /label=T2A

 CDS 5589..6305

 /label=BFP

 CDS 6312..6368

 /label=P2A

 CDS 6375..7826

 /label=E1b55k

/label=Ad4E1B

 polyA_signal 7827..8058

 /label=pGHpA

/label=bpA

 CDS 9130..9990

 /label=ampR

ORIGIN

 1 CCCCACGAGG GCCTATTTCC CATGATTCCT TCATATTTGC ATATACGATA CAAGGCTGTT

 61 AGAGAGATAA TTGGAATTAA TTTGACTGTA AACACAAAGA TATTAGTACA AAATACGTGA

 121 CGTAGAAAGT AATAATTTCT TGGGTAGTTT GCAGTTTTAA AATTATGTTT TAAAATGGAC

 181 TATCATATGC TTACCGTAAC TTGAAAGTAT TTCGATTTCT TGGCTTTATA TATCTTGTGG

 241 AAAGGACGAA ACACCGGGTC TTCGAGAAGA CCTGTTTTAG AGCTAGAAAT AGCAAGTTAA

 301 AATAAGGCTA GTCCGTTATC AACTTGAAAA AGTGGCACCG AGTCGGTGCT TTTTTGTTTT

 361 AGAGCTAGAA ATAGCAAGTT AAAATAAGGC TAGTCCGTTT TTAGCGCGTG CGCCAATTCT

 421 GCAGACAAAT GGCTCTAGAG GTACCCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT

 481 GACCGCCCAA CGACCCCCGC CCATTGACGT CAATAGTAAC GCCAATAGGG ACTTTCCATT

 541 GACGTCAATG GGTGGAGTAT TTACGGTAAA CTGCCCACTT GGCAGTACAT CAAGTGTATC

 601 ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTGTG

 661 CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG

 721 CTATTACCAT GGTCGAGGTG AGCCCCACGT TCTGCTTCAC TCTCCCCATC TCCCCCCCCT

 781 CCCCACCCCC AATTTTGTAT TTATTTATTT TTTAATTATT TTGTGCAGCG ATGGGGGCGG

 841 GGGGGGGGGG GGGGCGCGCG CCAGGCGGGG CGGGGCGGGG CGAGGGGCGG GGCGGGGCGA

 901 GGCGGAGAGG TGCGGCGGCA GCCAATCAGA GCGGCGCGCT CCGAAAGTTT CCTTTTATGG

 961 CGAGGCGGCG GCGGCGGCGG CCCTATAAAA AGCGAAGCGC GCGGCGGGCG GGAGTCGCTG

 1021 CGACGCTGCC TTCGCCCCGT GCCCCGCTCC GCCGCCGCCT CGCGCCGCCC GCCCCGGCTC

 1081 TGACTGACCG CGTTACTCCC ACAGGTGAGC GGGCGGGACG GCCCTTCTCC TCCGGGCTGT

 1141 AATTAGCTGA GCAAGAGGTA AGGGTTTAAG GGATGGTTGG TTGGTGGGGT ATTAATGTTT

 1201 AATTACCTGG AGCACCTGCC TGAAATCACT TTTTTTCAGG TTGGACCGGT GCCACCATGG

 1261 ACTATAAGGA CCACGACGGA GACTACAAGG ATCATGATAT TGATTACAAA GACGATGACG

 1321 ATAAGATGGC CCCAAAGAAG AAGCGGAAGG TCGGTATCCA CGGAGTCCCA GCAGCCGACA

 1381 AGAAGTACAG CATCGGCCTG GACATCGGCA CCAACTCTGTGGGCTGGGCC GTGATCACCG

 1441 ACGAGTACAA GGTGCCCAGC AAGAAATTCA AGGTGCTGGG CAACACCGAC CGGCACAGCA

 1501 TCAAGAAGAA CCTGATCGGA GCCCTGCTGT TCGACAGCGG CGAAACAGCC GAGGCCACCC

 1561 GGCTGAAGAG AACCGCCAGA AGAAGATACA CCAGACGGAA GAACCGGATC TGCTATCTGC  1621 AAGAGATCTT CAGCAACGAG ATGGCCAAGG TGGACGACAG CTTCTTCCAC AGACTGGAAG

 1681 AGTCCTTCCT GGTGGAAGAG GATAAGAAGC ACGAGCGGCA CCCCATCTTC GGCAACATCG

 1741 TGGACGAGGT GGCCTACCAC GAGAAGTACC CCACCATCTA CCACCTGAGA AAGAAACTGG

 1801 TGGACAGCAC CGACAAGGCC GACCTGCGGC TGATCTATCT GGCCCTGGCC CACATGATCA

 1861 AGTTCCGGGG CCACTTCCTG ATCGAGGGCG ACCTGAACCC CGACAACAGC GACGTGGACA

 1921 AGCTGTTCAT CCAGCTGGTG CAGACCTACA ACCAGCTGTT CGAGGAAAAC CCCATCAACG

 1981 CCAGCGGCGT GGACGCCAAG GCCATCCTGT CTGCCAGACT GAGCAAGAGC AGACGGCTGG

 2041 AAAATCTGAT CGCCCAGCTG CCCGGCGAGA AGAAGAATGG CCTGTTCGGA AACCTGATTG

 2101 CCCTGAGCCT GGGCCTGACC CCCAACTTCA AGAGCAACTT CGACCTGGCC GAGGATGCCA

 2161 AACTGCAGCT GAGCAAGGAC ACCTACGACG ACGACCTGGA CAACCTGCTG GCCCAGATCG

 2221 GCGACCAGTA CGCCGACCTG TTTCTGGCCG CCAAGAACCT GTCCGACGCC ATCCTGCTGA

 2281 GCGACATCCT GAGAGTGAAC ACCGAGATCA CCAAGGCCCC CCTGAGCGCC TCTATGATCA

 2341 AGAGATACGA CGAGCACCAC CAGGACCTGA CCCTGCTGAA AGCTCTCGTG CGGCAGCAGC

 2401 TGCCTGAGAA GTACAAAGAG ATTTTCTTCG ACCAGAGCAA GAACGGCTAC GCCGGCTACA

 2461 TTGACGGCGG AGCCAGCCAG GAAGAGTTCT ACAAGTTCAT CAAGCCCATC CTGGAAAAGA

 2521 TGGACGGCAC CGAGGAACTG CTCGTGAAGC TGAACAGAGA GGACCTGCTG CGGAAGCAGC

 2581 GGACCTTCGA CAACGGCAGC ATCCCCCACC AGATCCACCT GGGAGAGCTG CACGCCATTC

 2641 TGCGGCGGCA GGAAGATTTT TACCCATTCC TGAAGGACAA CCGGGAAAAG ATCGAGAAGA

 2701 TCCTGACCTT CCGCATCCCC TACTACGTGG GCCCTCTGGC CAGGGGAAAC AGCAGATTCG

 2761 CCTGGATGAC CAGAAAGAGC GAGGAAACCA TCACCCCCTG GAACTTCGAG GAAGTGGTGG

 2821 ACAAGGGCGC TTCCGCCCAG AGCTTCATCG AGCGGATGAC CAACTTCGAT AAGAACCTGC

 2881 CCAACGAGAA GGTGCTGCCC AAGCACAGCC TGCTGTACGA GTACTTCACC GTGTATAACG

 2941 AGCTGACCAA AGTGAAATAC GTGACCGAGG GAATGAGAAA GCCCGCCTTC CTGAGCGGCG

 3001 AGCAGAAAAA GGCCATCGTG GACCTGCTGT TCAAGACCAA CCGGAAAGTG ACCGTGAAGC

 3061 AGCTGAAAGA GGACTACTTC AAGAAAATCG AGTGCTTCGA CTCCGTGGAA ATCTCCGGCG

 3121 TGGAAGATCG GTTCAACGCC TCCCTGGGCA CATACCACGA TCTGCTGAAA ATTATCAAGG

 3181 ACAAGGACTT CCTGGACAAT GAGGAAAACG AGGACATTCT GGAAGATATC GTGCTGACCC

 3241 TGACACTGTT TGAGGACAGA GAGATGATCG AGGAACGGCT GAAAACCTAT GCCCACCTGT

 3301 TCGACGACAA AGTGATGAAG CAGCTGAAGC GGCGGAGATA CACCGGCTGG GGCAGGCTGA

 3361 GCCGGAAGCT GATCAACGGC ATCCGGGACA AGCAGTCCGG CAAGACAATC CTGGATTTCC

 3421 TGAAGTCCGA CGGCTTCGCC AACAGAAACT TCATGCAGCT GATCCACGAC GACAGCCTGA

 3481 CCTTTAAAGA GGACATCCAG AAAGCCCAGG TGTCCGGCCA GGGCGATAGC CTGCACGAGC

 3541 ACATTGCCAA TCTGGCCGGC AGCCCCGCCA TTAAGAAGGG CATCCTGCAG ACAGTGAAGG

TUNEology 波长可调检测卡盒-->