LOCUS pLEW82 5446 bp DNA circular 14-JUL-2009 pJEL61 is a pLew82 derivative made* by Joanna Lowell for fusing eYFP (Clontech) to the C-terminus of inserts at one of the unique sites in the linker region. Unlike many of our other fusion cloning vectors, this one contains multiple cloning sites. *Cut pEYFP-1 with Not I, filled site with klenow, and cut with HindIII to release eYFP fragment. Ligated this fragment into pLEW82 opened with Bam HI (filled with klenow) and Hind III. FEATURES Location/Qualifiers misc_feature complement(1..713) /function="rRNA Spacer" promoter 720..739 /function="T7 Promoter" /note="T7 promoter & initiation region. Promoter-Op-Luc cassette orig from Lew19" 5'UTR 770..825 /function="GPEET upstream" /note="to splice site. Note that this is truncated at the upstream SmaI site compared to the GPEET upstream region in pLew20" 5'UTR 826..856 /function="GPEET 5´ UTR" 3'UTR 1640..1787 /function="ALD 'short' 3´ UTR" /note="Perfect match with 3480 to 3612 in X52586" /note="truncated aldolase 3'UTR originally derived from pHD330 (about 6x lower BF expression than the full-length ALD UTR in pLew100, but no difference in expression levels in PF, between long and short UTRs) " 5'UTR 1793..1907 /function="ACT 5´ UTR" /note="actin-derived sas; used in all HD330, 430 & Lew5 descendants" CDS 1917..2288 /function="BLE" 3'UTR 2343..2630 /function="ACT 3´ UTR" /note="actin 3'UTR- same as what's used in all HD330, 430 and Lew5 descendants" terminator 2637..2769 /function="T7 Term" /note="132bp Pet3a fragment (originally PCR'd), then subcloned from published HD216, bearing T7 terminator" terminator 2770..2902 /function="T7 Term" /note="132 bp Pet3a fragment (originally PCR'd then subcloned from published HD216) bearing T7 terminator" CDS 3591..4448 /function="LACTAMASE" conflict 766..773 /note="From a restriction digest of a pLew111 derivative, an SrfI site (GCCCGGGC) originally designated here was absent, according to Shawn Motyka (Barbara Sollner Webb lab) email 9/8/3. This was confirmed by George from 2 seq trace files of Fabian's pLew111-bsd construct." protein_bind 745..763 /function="Tet Operator" misc_feature 857..912 /function="Linker fusion peptide" CDS 913..1632 /function="eYFP" /note="Cut Clontech pEYFP-1 with Not I, filled site with klenow, and cut with Hind III to release eYFP fragment. Ligated this fragment into pLEW82 opened with Bam HI (filled with klenow) and Hind III." BASE COUNT 1315 a 1432 c 1457 g 1242 t ORIGIN 1 GAATTCGAGC TCATATAGTT GGTATGTATT CTAATTCCAG ACTACTGGCG TGGATAAACA 61 TGTCCCCTGA TTAAAAGGAA AGATTCCATA GCCCATTAGT GCAAAGATAA TTGGTACACC 121 GTCAAAAACA CATGGGAGAC CACACGATAC GACACTGTGA CCGTAGCATC AATTCCGCAC 181 TCGATATAAC TCTATCGAAG AACTTCCATG GTACAAACTG GTCGCCACGG TCTGCTCCAC 241 ACGGAGATCA TCGTATCATT TTTATCGATA GCGGCCGCTA TCGATGTATG CCTTGGCCCT 301 GATGGCATGC CAATTTCACT ACAACGGACT ATGTGGACCC CGTTATCATG GAAATGCGCT 361 AGTTGGAGGA AGTTAGACCG CGCCGGAAAA GAGAGGGGTA GAGAAAATGA CAACTTGGAA 421 GATATCCACA CACGCACGGT GAAACGTTAG CAACAATTAT TAGGGAAGCG ACGCTTGCCC 481 TAGTCCCACG AGTAACCAAG ACTCCAAAAG CCTTTCTGGC ACAGAGAGCG AGCCGAAATG 541 GAAAAGAGAA ACAATGCCTG CACTAACACT ACTGAGCGAT TCGCCTCGCC GCGGAGGACC 601 GAATACTAAT AACGACACTT GCGGTCAAAA AGTAGAAGAA CAAATGCTCA ACGATGAGTG 661 AATCAGGTTA GGGTAGTTGG AAAATTATAC AGAATGTCTT TGGCAACACA CCGGCTGATT 721 AATACGACTC ACTATAGGGA GATCTCCCTA TCAGTGATAG AGATCTCCCG GGCTGCACGC 781 GCCTTCGAGT TTTTTTTCCT TTTCCCCATT TTTTTCAACT TGAAGACTTC AATTACACCA 841 AAAAGTAAAA TTCACAAGCT TCGAATTCTG CAGTCGACGG TACCGCGGGC CCGGGATCCA 901 CCGGTCGCCA CCATGGTGAG CAAGGGCGAG GAGCTGTTCA CCGGGGTGGT GCCCATCCTG 961 GTCGAGCTGG ACGGCGACGT AAACGGCCAC AAGTTCAGCG TGTCCGGCGA GGGCGAGGGC 1021 GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCCGTG 1081 CCCTGGCCCA CCCTCGTGAC CACCTTCGGC TACGGCCTGC AGTGCTTCGC CCGCTACCCC 1141 GACCACATGA AGCAGCACGA CTTCTTCAAG TCCGCCATGC CCGAAGGCTA CGTCCAGGAG 1201 CGCACCATCT TCTTCAAGGA CGACGGCAAC TACAAGACCC GCGCCGAGGT GAAGTTCGAG 1261 GGCGACACCC TGGTGAACCG CATCGAGCTG AAGGGCATCG ACTTCAAGGA GGACGGCAAC 1321 ATCCTGGGGC ACAAGCTGGA GTACAACTAC AACAGCCACA ACGTCTATAT CATGGCCGAC 1381 AAGCAGAAGA ACGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGA GGACGGCAGC 1441 GTGCAGCTCG CCGACCACTA CCAGCAGAAC ACCCCCATCG GCGACGGCCC CGTGCTGCTG 1501 CCCGACAACC ACTACCTGAG CTACCAGTCC GCCCTGAGCA AAGACCCCAA CGAGAAGCGC 1561 GATCACATGG TCCTGCTGGA GTTCGTGACC GCCGCCGGGA TCACTCTCGG CATGGACGAG 1621 CTGTACAAGT AAAGCGGCCG ATCCTGCCCA TTTAGTTGGC TTTTCCCTTG TCTCGTGTCT 1681 TTTCCGTGGA AAGGTTCCCG GAGTAATCTG ATGGCACAGC AGGGAGGTGC GCCTGCAGGT 1741 TGGTTAGGAA GGGGGGATGA TGTAAAAGAA GAAAATGGGG GGATTCGAGC CCGGGCACAG 1801 CAAGGTCTTC TGAAATTCAT GTTTTTTTTT TTTTTACTCT GCATTGCAGT CTCCGCTCTT 1861 ATTTAGTTTT GCTTTACGTA AGGTCTCGTT GCTGCCATAA AATAAGCTAT ATCACCATGG 1921 CCAAGTTGAC CAGTGCCGTT CCGGTGCTCA CCGCGCGCGA CGTCGCCGGA GCGGTCGAGT 1981 TCTGGACCGA CCGGCTCGGG TTCTCCCGGG ACTTCGTGGA GGACGACTTC GCCGGTGTGG 2041 TCCGGGACGA CGTGACCCTG TTCATCAGCG CGGTCCAGGA CCAGGTGGTG CCGGACAACA 2101 CCCTGGCCTG GGTGTGGGTG CGCGGCCTGG ACGAGCTGTA CGCCGAGTGG TCGGAGGTCG 2161 TGTCCACGAA CTTCCGGGAC GCCTCCGGGC CGGCCATGAC CGAGATCGGC GAGCAGCCGT 2221 GGGGGCGGGA GTTCGCCCTG CGCGACCCGG CCGGCAACTG CGTGCACTTC GTGGCCGAGG 2281 AGCAGGACTG ACCGACGCCG ACCAACACCG CCGGTCCGAC GGCGGCCCAC GGGTCCCAGG 2341 CCTCGGAGAT CCTAACACCG GGTTGTGTGG CCAAAATTGT TCTGTAGTCG CTGTGAGTTG 2401 ACACGGCTAG TGCTTATGAT TTTCCTCGCG TGTGGTGCCT GTACTCAGCC CTATGCCTTA 2461 TTTGCAACAC ATTTACGTAC AGCGCACAAG AGAAGAGAAG ATCACTTGAA GATAATAAAT 2521 ATAGGGTTGT AGGCATCTTG TTTAACTCAA ATTTTCTCGT CTTGGTGTGT CGACATGATT 2581 GAAATAGTGC CACCAGTTGT GTTTGATGCG TTTGTTATCT ATGCAGTATT CTGCAGGCTG 2641 CTAACAAAGC CCGAAAGGAA GCTGAGTTGG CTGCTGCCAC CGCTGAGCAA TAACTAGCAT 2701 AACCCCTTGG GGCCTCTAAA CGGGTCTTGA GGGGTTTTTT GCTGAAAGGA GGAACTATAT 2761 CCGGATAGGG CTGCTAACAA AGCCCGAAAG GAAGCTGAGT TGGCTGCTGC CACCGCTGAG 2821 CAATAACTAG CATAACCCCT TGGGGCCTCT AAACGGGTCT TGAGGGGTTT TTTGCTGAAA 2881 GGAGGAACTA TATCCGGATA GGGCCCCTGC AGGCATGCAA GCTAGCCAGA TCCTCTACGC 2941 CGGACGCATC GTGGCCGGCA TCACCGGCGC CACAGGTGCG GTTGCTGGCG CCTATATCGC 3001 CGACATCACC GATGGGGAAG ATCGGGCTCG CCACTTCGGG CTCATGAGCG CTTGTTTCGG 3061 CGTGGGTATG GTGGCAGGCC CCGTGGCCGG GGGACTGTTG GGCGCCATCT CCTTGCACCA 3121 TTCCTTGCGG CGGCGGTGCT CAACGGCCTC AACCTACTAC TGGGCTGCTT CCTAATGCAG 3181 GAGTCGCATA AGGGAGAGCG TCGATATGGT GCACTCTCAG TACAATCTGC TCTGATGCCG 3241 CATAGTTAAG CCAGCCCCGA CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC 3301 TGCTCCCGGC ATCCGCTTAC AGACAAGCTG TGACCGTCTC CGGGAGCTGC ATGTGTCAGA 3361 GGTTTTCACC GTCATCACCG AAACGCGCGA GACGAAAGGG CCTCGTGATA CGCCTATTTT 3421 TATAGGTTAA TGTCATGATA ATAATGGTTT CTTAGACGTC AGGTGGCACT TTTCGGGGAA 3481 ATGTGCGCGG AACCCCTATT TGTTTATTTT TCTAAATACA TTCAAATATG TATCCGCTCA 3541 TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA AAGGAAGAGT ATGAGTATTC 3601 AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT GTTTTTGCTC 3661 ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA CGAGTGGGTT 3721 ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC GAAGAACGTT 3781 TTCCAATGAT GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC CGTATTGACG 3841 CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG GTTGAGTACT 3901 CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA TGCAGTGCTG 3961 CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC GGAGGACCGA 4021 AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT GATCGTTGGG 4081 AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG CCTGTAGCAA 4141 TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT TCCCGGCAAC 4201 AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC TCGGCCCTTC 4261 CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT CGCGGTATCA 4321 TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC ACGACGGGGA 4381 GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC TCACTGATTA 4441 AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT TTAGATTGAT TTAAAACTTC 4501 ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA TAATCTCATG ACCAAAATCC 4561 CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT AGAAAAGATC AAAGGATCTT 4621 CTTGAGATCC TTTTTTTCTG CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC 4681 CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT 4741 TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTA GGCCACCACT 4801 TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG 4861 CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA 4921 AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA 4981 CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA AAGCGCCACG CTTCCCGAAG 5041 GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG 5101 AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT CGGGTTTCGC CACCTCTGAC 5161 TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA 5221 ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG 5281 CGTTATCCCC TGATTCTGTG GATAACCGTA TTACCGCCTT TGAGTGAGCT GATACCGCTC 5341 GCCGCAGCCG AACGACCGAG CGCAGCGAGT CAGTGAGCGA GGAAGCGGAA GAGCGCCCAA 5401 TACGCAAACC GCCTCTCCCC GCGCGTTGGC CGATTCATTA ATGCAG //