pGreenPuro shRNA vector慢病毒shRNA沉默干扰载体GFP

Product Data Sheet

Recovery

1. 1.   Obtain an LB agar plate with the appropriate antibiotic.

2.Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.)

3.Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1.

1. 4.  Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.

5.Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.

6.Grow overnight in a 37 ^o C incubator (unless a different growth temperature is indicated on the plasmid datasheet).

1. 7.  In the morning, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies.

Sequence:

LOCUS pGreenPuro (pSIH1_H1_copGFP_T2A_Puro)

7861 bp ds-DNA circular

31-DEC-2008

FEATURES Location/Qualifiers

misc_feature 1921..2270

/label=CMV

/ApEinfo_fwdcolor=cyan

/ApEinfo_revcolor=green

promoter 7..234

/vntifkey="29"

/label=RSV\promoter

/ApEinfo_fwdcolor=pink

/ApEinfo_revcolor=pink

misc_feature 3041..3094

/label=T2A

/ApEinfo_fwdcolor=#ff80ff

/ApEinfo_revcolor=green

misc_feature complement(5579..6252)

/vntifkey="21"

/label=pUC\ORI

/ApEinfo_fwdcolor=#0000ff

/ApEinfo_revcolor=#8000ff

misc_feature 3095..3694

/label=Puro

/ApEinfo_fwdcolor=#ff00ff

/ApEinfo_revcolor=green

CDS complement(6397..7257)

/vntifkey="4"

/label=AmpR

/ApEinfo_fwdcolor=pink

/ApEinfo_revcolor=pink

misc_feature 1798..1915

/vntifkey="21"

/label=cPPT

/ApEinfo_fwdcolor=#ffff80

/ApEinfo_revcolor=#8000ff

misc_feature 4610..4615

/label=BamH I

/ApEinfo_fwdcolor=cyan

/ApEinfo_revcolor=green

misc_feature 4619..4624

/label=EcoRI

/ApEinfo_fwdcolor=cyan

/ApEinfo_revcolor=green

misc_feature 2285..3040

/label=copGFP

/ApEinfo_fwdcolor=#00ff00

/ApEinfo_revcolor=green

LTR 235..414

/vntifkey="19"

/label=HIV\LTR

/ApEinfo_fwdcolor=pink

/ApEinfo_revcolor=pink

misc_feature 567..919

/vntifkey="21"

/label=Gag

/ApEinfo_fwdcolor=#ff00ff

/ApEinfo_revcolor=#8000ff

misc_feature 1076..1308

/vntifkey="21"

/label=RRE

/ApEinfo_fwdcolor=#ffff00

/ApEinfo_revcolor=#8000ff

misc_feature 1309..1797

/vntifkey="21"

/label=env

/ApEinfo_fwdcolor=#0000ff

/ApEinfo_revcolor=#8000ff

misc_feature join(3695..4229,4231..4233,4235..4283)

/label=WPRE

/ApEinfo_fwdcolor=cyan

/ApEinfo_revcolor=green

misc_feature 4517..4607

/label=H1 promoter

/ApEinfo_fwdcolor=#ff00ff

/ApEinfo_revcolor=green

misc_feature 4522..4545

/label=FGNF sequencing primer

/ApEinfo_fwdcolor=#ffff00

/ApEinfo_revcolor=green

misc_feature 4902..5209

/label=SV40 ori

/ApEinfo_fwdcolor=cyan

/ApEinfo_revcolor=green

ORIGIN

1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca

61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta

121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga

181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc

241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta

301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact

361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg

421 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct

481 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt

541 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag

601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt

661 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt

721 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg

781 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag

841 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag

901 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg

961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag

1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag

1081 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc

1141 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga

1201 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc

1261 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg

1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata

1381 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa

1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga

1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa

1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat

1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt

1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg

1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggttaacttt

1801 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc

1861 aacagacata caaactaaag aattacaaaa acaaattaca aaattcaaaa ttttatcgat

1921 actagtatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg

1981 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat

2041 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt

2101 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc

2161 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc

2221 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga ttctagaCGC

2281 CACCatggag agcgacgaga gcggcctgcc cgccatggag atcgagtgcc gcatcaccgg

2341 caccctgaac ggcgtggagt tcgagctggt gggcggcgga gagggcaccc ccaagcaggg

2401 ccgcatgacc aacaagatga agagcaccaa aggcgccctg accttcagcc cctacctgct

2461 gagccacgtg atgggctacg gcttctacca cttcggcacc taccccagcg gctacgagaa

2521 ccccttcctg cacgccatca acaacggcgg ctacaccaac acccgcatcg agaagtacga

2581 ggacggcggc gtgctgcacg tgagcttcag ctaccgctgc gaggccggcc gcgtgatcgg

2641 cgacttcaag gtggtgggca ccggcttccc cgaggacagc gtgatcttca ccgacaagat

2701 catccgcagc aacgccaccg tggagcacct gcaccccatg ggcgataacg tgctggtggg

2761 cagcttcgcc cgcaccttca gcctgcgcga cggcggctac cacagcttcg tggtggacaa

2821 ccacatgcac ttcaagagcg ccatccaccc cagcatcctg cagaacgggg gccccatgtt

2881 cgccttccgc cgcgtggagg agctgcacag caacaccgag ctgggcatcg tggagtacca

2941 gcacgccttc aagaccccca tcgccttcgc cagatcccgc gctcagtcgt ccaattctgc

3001 cgtggacggc accgccggac ccggctccac cggatctcgc GAGGGCAGAG

GAAGTCTTCT

3061 AACATGCGGT GACGTGGAGG AGAATCCCGG CCCTatgacc gagtacaagc

ccacggtgcg

3121 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga

3181 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct

3241 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga