d0d0=ID EU424172; SV 1; circular; other DNA; STD; SYN; 8254 BP. AC EU424172; DT 03-FEB-2009 (Rel. 99, Created) DT 03-FEB-2009 (Rel. 99, Last updated, Version 1) DE Cloning vector L071 RRI-Green, complete sequence. KW . OS Cloning vector L071 RRI-Green OC other sequences; artificial sequences; vectors. RN [1] RP 1-8254 RA Micklem D.R., Lorens J.B.; RT "Retroviral vectors for expression of shRNAs"; RL Unpublished. RN [2] RP 1-8254 RA Micklem D.R., Lorens J.B.; RT ; RL Submitted (24-JAN-2008) to the EMBL/GenBank/DDBJ databases. RL Biomedicine, University of Bergen, Jonas Lies vei 91, Bergen 5009, Norway FH Key Location/Qualifiers FT source 1..8254 FT /organism="Cloning vector L071 RRI-Green" FT /mol_type="other DNA" FT /db_xref="taxon:517185" FT LTR 2..747 FT /note="CRU5 chimeric CMV promoter, MoMLV and MSCV 5' LTR" FT enhancer 2..571 FT /note="CMV immediate-early enhancer region" FT misc_feature 602..669 FT /note="R region" FT misc_feature 748..1665 FT /note="psi+; chimeric MoMLV/MSCV extended packaging signal" FT misc_feature complement(2063..2270) FT /note="BGHpA signal; bovine growth hormone polyadenylation FT signal" FT mRNA complement(join(2157..3742,4666..4698)) FT /product="GFP2APuro fusion protein" FT polyA_site complement(2157) FT CDS complement(2290..3708) FT /codon_start=1 FT /transl_table=11 FT /product="GFP2APuro fusion protein" FT /note="fusion between EGFP, FMDV 2A-related self-cleaving FT peptide and puromycin acetyl transferase; selectable marker FT conferring green fluorescence and resistance to puromycin" FT /protein_id="ACB70965.1" FT /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL FT KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD FT GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK FT VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL FT EFVTAAGITLGMDELYKEFGGGSGGGQLLNFDLLKLAGDVESNPGPTTSMTEYKPTVRL FT ATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADD FT GAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATV FT GVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEG FT PRDLVHDPQARCLI" FT misc_feature complement(2901..2985) FT /note="FMDV 2A self-cleaving linker region" FT promoter complement(3734..4901) FT /note="EF1-alpha (elongation factor 1 alpha) promoter and FT first intron" FT intron complement(3743..4665) FT /note="EF1-alpha intron" FT misc_difference complement(4543..4589) FT /note="not identical to canonical EF1-alpha intron FT sequence" FT TATA_signal complement(4722..4728) FT LTR 4962..5869 FT /note="SIN self-inactivating LTR with U6 expression FT cassette" FT promoter 5089..5354 FT /note="modified human U6 promoter" FT misc_binding 5307..5324 FT /bound_moiety="Tet repressor" FT /note="TetO binding site" FT misc_feature 5355..5745 FT /note="stuffer fragment; lost during cloning of shRNAs FT between unique BglII and AarI sites" FT misc_feature 5355 FT /note="U6 promoter-dependent transcription initiation" FT misc_feature 5802..5869 FT /note="R region" FT rep_origin complement(6418..7130) FT /note="pMB1 ori (pUC)" FT gene complement(7195..8055) FT /gene="bla" FT CDS complement(7195..8055) FT /codon_start=1 FT /transl_table=11 FT /gene="bla" FT /product="bla" FT /note="beta-lactamase; ampicillin resistance" FT /protein_id="ACB70964.1" FT /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI FT ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS FT PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW FT EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA FT LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS FT LIKHW" SQ Sequence 8254 BP; 1867 A; 2325 C; 2166 G; 1896 T; 0 other; cctagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 60 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 120 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 180 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 240 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 300 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 360 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg 420 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 480 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcat 540 gtacggtggg aggtctatat aagcagagct caataaaaga gcccacaacc cctcactcgg 600 cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat aaagcctctt 660 gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc agattgattg 720 actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag acccctgccc 780 agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc gtgtctgtct 840 ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg tactagttag 900 ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttcgga acacccggcc 960 gcaaccctgg gagacgtccc agggacttcg ggggccgttt ttgtggcccg acctgagtcc 1020 aaaaatcccg atcgttttgg actctttggt gcacccccct tagaggaggg atatgtggtt 1080 ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt 1140 tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt 1200 ctgactgtgt ttctgtattt gtctgaaaat atgggcccgg gccagactgt taccactccc 1260 ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa ccagtcggta 1320 gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac ctttaacgtc 1380 ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa gatcaaggtc 1440 ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac ctgggaagcc 1500 ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc tccgcctcct 1560 cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc gcctcgatcc 1620 tccctttatc cagccctcac tccttctcta ggcgccccca tatggccata tgagatcgat 1680 cttatatggg gcacccccgc cccttgtaaa cttccctgac cctgacatga caagagttac 1740 taacagcccc tctctccaag ctcacttaca ggctctctac ttagtccagc acgaagtctg 1800 gagacctctg gcggcagcct accaagaaca actggaccga ccggtggtac ctcaccctta 1860 ccgagtcggc gacacagtgt gggtccgccg acaccagact aagaacctag aacctcgctg 1920 gaaaggacct tacacagtcc tgctgaccac ccccaccgcc ctcaaagtag acggcatcgc 1980 agcttggata cacgccgccc acgtgaaggc tgccgacccc gggggtggac catcctctag 2040 actgccggat ctcgaaggat cttccccacc atgcctgcta ttgtcttccc aatcctcccc 2100 cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga 2160 tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga 2220 aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcgaggtcta 2280 gcggccgctc tagatcaggc accgggcttg cgggtcatgc accaggtcgc gcggtccttc 2340 gggcacctcg acgtcggcgg tgacggtgaa gccgagccgc tcgtagaagg ggaggttgcg 2400 gggcgcggag gtctccagga aggcgggcac cccggcgcgc tcggccgcct ccactccggg 2460 gagcacgacg gcgctgccca gacccttgcc ctggtggtcg ggcgagacgc cgacggtggc 2520 caggaaccac gcgggctcct tgggccggtg cggcgccagg aggccttcca tctgttgctg 2580 cgcggccagc cgggaaccgc tcaactcggc catgcgcgga ccgatctcgg cgaacaccgc 2640 ccccgcttcg acgctctccg gcgtggtcca gaccgccacc gcggcgccgt cgtccgcgac 2700 ccacaccttg ccgatgtcga gcccgacgcg cgtgaggaag agttcttgca gctcggtgac 2760 ccgctcgatg tggcggtccg ggtcgacggt gtggcgcgtg gcggggtagt cggcgaacgc 2820 ggcggcgagg gtgcgtacgg cccgggggac gtcgtcgcgg gtggcgaggc gcaccgtggg 2880 cttgtactct gtcattgatg tggtgggccc agggttggac tcgacgtctc ccgcaagttt 2940 aagaaggtca aaattcaaca gctgaccgcc accgctgcca cctccgaatt ccttgtacag 3000 ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc 3060 gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg 3120 cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac 3180 gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt 3240 gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat 3300 gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc 3360 ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg 3420 ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc 3480 ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg 3540 cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc 3600 gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac 3660 caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgg tggatcgatc 3720 ctaggccacg tgttcacgac acctgaaatg gaagaaaaaa actttgaacc actgtctgag 3780 gcttgagaat gaaccaagat ccaaactcaa aaaggccaaa ttccaaggag aattacatca 3840 agtgccaagc tggcctaact tcagtctcca cccactcagt gtggggaaac tccatcgcat 3900 aaaacccctc cccccaacct aaagacgacg tactccaaaa gctccagaac taatcgaggt 3960 gcctggacgg cgcccggtac tccgtggagt cacatgaagc gacggctgag gacggaaagg 4020 cccctttcct ttgtgtgggt gactcacccg cccgctctcc cgagcgccgc gtcctccatt 4080 ttgagccccc tggagcaggg ccgggaagcg gccatctttc cgctcacgca actggtgccg 4140 accgggccag ccttgccgcc cagggcgggg cgatacacgg cggcgcgagg ccaggcacca 4200 gagcaggccg gccagcttga gactaccccc gtccgattct cggtggccgc gctcgcaggc 4260 cccgcctcgc cgaacatgtg cgctgggacg cacgggcccc gtcgccgccc gcggccccaa 4320 aaaccgaaat accagtgtgc agatcctggc ccgcatttac aagactatct tgccagaaaa 4380 aaagcgtcgc agcaggtcat caaaaatttt aaatggctag agacttatcg aaagcagcga 4440 gacaggcgcg aaggtgccac cagattcgca cgcggcggcc ccagcgccca ggccaggcct 4500 caactcaagc acgaggcgaa ggggctccta aagcgcaagg cccgcccctg gctccagctc 4560 gggatcaaga atcacgtact ggagccaggt ggaagtaatt caaggcacgc aagggccata 4620 acccgtaaag aggccaggcc cgcgggaacc acacacggca cttacctgtg ttctggcggc 4680 aaacccgttg cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc 4740 accctcggga aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca 4800 ccttctctag gcaccggttc aattgccgac ccctcccccc aacttctcgg ggactgtggg 4860 cgatgtgcgc tctgcccact gacgggcacc ggagcctcac gctcgagagg ccgctcgacg 4920 ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt 4980 aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg 5040 agaatagaaa agttcagatc aaggtcagga acagaggaac aggtcgacca aggtcgggca 5100 ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag 5160 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg 5220 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta 5280 tcatatgctt accgtaactt gaaagtactc tatcattgat agagttatat atcttgtgga 5340 aaggacagat ctccgcatgc ggatccagga acaatggcag ctttcttgcc atcaaagctg 5400 ctgatggcac atatattctt aatggtgact acactttgtc caccttagag caagacatta 5460 tgtacaaagg tgttgtcttg aggtacagcg gctcctctgc ggcattggaa agaattcgca 5520 gctttagccc tctcaaagag cccttgacca tccaggttct tactgtgggc aatgcccttc 5580 gacctaaaat taaatacacc tacttcgtaa agaagaagaa ggaatctttc aatgctatcc 5640 ccactttttc agcatgggtc attgaagagt ggggcgaatg ttctaagtca tgtgaattgg 5700 gttggcagag aagactggta gaatgccgag acatcacctg catgcttttt ccatactttt 5760 tttggcaagc ttacgcgtga gcccacaacc cctcactcgg ggcgccagtc ctccgattga 5820 ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg 5880 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct 5940 ttcacatgca gcatgtatca aaattaattt ggtttttttt cttaagtatt tacattaaat 6000 ggccatagta gttcattatg gacagcgcag aaagagctgg ggagaattgt gaaattgtta 6060 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 6120 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 6180 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 6240 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 6300 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 6360 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 6420 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 6480 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 6540 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 6600 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 6660 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 6720 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 6780 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 6840 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 6900 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 6960 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 7020 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 7080 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 7140 atgaagtttg cgcaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 7200 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 7260 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 7320 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 7380 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 7440 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 7500 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 7560 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 7620 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 7680 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 7740 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 7800 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 7860 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 7920 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 7980 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 8040 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 8100 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 8160 cacatttccc cgaaaagtgc caccagcttt gctcttagga gtttcctaat acatcccaaa 8220 ctcaaatata taaagcattt gacttgttct atgc 8254 > L071"Times New Roman d0ENdeI(237),NcoI(363),SacI(571),AscI(601),XmaI(630),SmaI(632),KpnI(638) ArialY> ,{1r5i-bi d0WSpeI(893),EagI(957),PstI(1178),XmaI(1237),ApaI(1238),SmaI(1239),Bsu36I(1273),PstI(1360) Arial> R d0BSexAI(1470),NdeI(1661),NdeI(1669),ClaI(1677),KpnI(1851),XmaI(2019) Arial>  d0MSmaI(2021),XbaI(2037),NotI(2283),EagI(2283),XbaI(2290),SexAI(2322),EagI(2443) Arial> mopF d0CSalI(2783),XmaI(2842),SmaI(2844),ApaI(2910),PvuII(2962),EcoRI(2987) Arial >  d0NcoI(3706),ClaI(3716) Arialz> #5CC[ d0ApaI Arial> .8AAM d0NXhoI(4903),NheI(4993),SalI(5084),NdeI(5284),ScaI(5307),BglII(5348),BamHI(5362) Arial> > d0EEcoRI(5513),HindIII(5768),MluI(5774),XmaI(5830),SmaI(5832),KpnI(5838) Arial> w d0PvuII Arial5> JJJDP d0ScaI ArialE> nvjmfcMWcd0> 2HpLXSN_5_primer"Times New Romand0C> 2H MSCV_primer"Times New Romand0C> XhpBABE_5_primer"Times New Romand0CɆ>  pBMN_5_primer"Times New Romand0C> R ORF 1"Times New Romand0C> BGH_rev_primer"Times New Romand0C>  BGHrev_primer"Times New Roman*d0CɆ> |ORF 2"Times New Romand0CɆ> |EGFP"Times New Romand0>  EGFP_C_primer"Times New Roman*d0CɆ> :O EGFP_N_primer"Times New Roman*d0C> EF1a_fwd_primer"Times New Romand0> AORF 2"Times New Romand0>  LKO1_5_primer"Times New Romand0C>  lac_promoter"Times New Romand0C> wbla"Times New Romand0C> wORF 1"Times New Roman(d0C>  AmpR_promoter"Times New Roman d0test"Times New Roman>  ' d0test"Times New Romanp > qPkY