LOCUS pET-29a(+) 5371 bp DNA circular 20-OCT-2003 DEFINITION T7 expression vector pET-29a(+). REFERENCE 1 (bases 1 to 5371) Novagen, Inc. pET-29a(+) vector sequence http://www.novagen.com/docs/NDIS/69871-000.html REFERENCE 2 (sites) Novagen, Inc. pET-29a-c(+) Vectors (TB076 12/98) http://www.novagen.com/docs/NDIS/TB076-000.pdf REFERENCE 3 (sites) Novagen, Inc. pET System Manual 10th Edition (TB055 07/02) http://www.novagen.com/docs/NDIS/TB055-000.pdf REFERENCE 4 (rep_origin endpoints) AUTHORS Selzer,G., Som,T., Itoh,T. and Tomizawa,J. TITLE The origin of replication of plasmid p15A and comparative studies on the nucleotide sequences around the origin of related plasmids JOURNAL Cell 32 (1), 119-129 (1983) MEDLINE 83129391 PUBMED 6186390 COMMENT Based on the T7 promoter-driven system originally developed by Studier and colleague, Novagen's pET System has been used to express thousands of different proteins. In pET vectors, target genes are cloned under control of strong bacteriophage T7 transcription and translation signals, and expression is induced by providing a source of T7 RNA polymerase in the host cell. The pET-29a-c(+) vectors carry an N-terminal S-Tag/thrombin configuration plus an optional C-terminal His-Tag sequence. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the cloning/expression region detail map. FEATURES Location/Qualifiers terminator complement(27..73) /note="T7 terminator" misc_feature 69..87 /note="T7 terminator primer #69337-3" misc_feature complement(140..157) /note="C-terminal His-tag coding sequence" /translation="HHHHHH" misc_feature complement(158..217) /note="multiple cloning region (NcoI-XhoI)" misc_feature complement(216..233) /note="thrombin cleavage site tag" /translation="LVPRGS" misc_feature complement(249..293) /note="N-terminal S-tag coding sequence" /translation="KETAAAKFERQHMDS" misc_feature complement(341..365) /note="lac operator" misc_feature complement(365..384) /note="T7 promoter primer #69348-3" promoter complement(368..384) /note="T7 promoter" misc_feature complement(415..430) /note="pET upstream primer #69214-3" CDS 775..1857 /gene="lacI" /product="transcriptional repressor of the lac operon" /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT QTASPRALADSLMQLARQVSRLESGQ" rep_origin complement(3287..3875) /standard_name="pMB1 (ColE1-like) plasmid origin of replication" /note="coordinates include the region from the RNA/DNA switch point to the -35 promoter sequence of the RNAII (primer) transcript" /direction=left CDS 3997..4812 /note="kanamycin resistance" /product="aminoglycoside 3'-phosphotransferase" /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" rep_origin complement(4905..5360) /standard_name="phage f1 origin of replication" /note="allows production of single stranded DNA upon infection with a helper phage" /direction=left BASE COUNT 1272 a 1387 c 1443 g 1269 t ORIGIN 1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 181 cgacggagct cgaattcgga tccgatatca gccatggaac cgcgtggcac cagggtaccc 241 agatctgggc tgtccatgtg ctggcgttcg aatttagcag cagcggtttc tttcatatgt 301 atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca 361 attcccctat agtgagtcgt attaatttcg cgggatcgag atcgatctcg atcctctacg 421 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg 481 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg 541 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg 601 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa 661 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc 721 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa 781 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc 841 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg 901 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg 961 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg 1021 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc 1081 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg 1141 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat 1201 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc 1261 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc 1321 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat 1381 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc 1441 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg 1501 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg 1561 cgcgttggtg cggacatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat 1621 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac 1681 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca 1741 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 1801 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 1861 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt 1921 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc 1981 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt 2041 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt 2101 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt 2161 cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct 2221 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc 2281 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac 2341 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc 2401 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc 2461 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg 2521 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc 2581 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag 2641 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc 2701 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga 2761 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg 2821 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 2881 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 2941 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac 3001 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat atgcggtgtg 3061 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 3121 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3181 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3241 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3301 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3361 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3421 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3481 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3541 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3601 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3661 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3721 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3781 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3841 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 3901 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa 3961 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg 4021 tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg 4081 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat 4141 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag 4201 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc 4261 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag 4321 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg 4381 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt 4441 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac 4501 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc 4561 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag 4621 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat 4681 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt 4741 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat 4801 gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa 4861 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaaattg taaacgttaa 4921 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc 4981 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt 5041 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa 5101 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg 5161 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg 5221 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc 5281 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa 5341 tgcgccgcta cagggcgcgt cccattcgcc a //