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Template	LOCUS U01668 4133 bp DNA circular SYN 01-JUL-1997 DEFINITION Phagemid cloning vector pKSS, complete sequence. ACCESSION U01668 VERSION U01668.1 GI:405986 KEYWORDS direct-selection cloning vehicle; positive-selection; pheS. SOURCE unidentified cloning vector. ORGANISM unidentified cloning vector artificial sequence; vectors. REFERENCE 1 (bases 3084 to 3868) 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, 119-129 (1983) MEDLINE 83129391 REFERENCE 2 (bases 59 to 1239) AUTHORS Fayat,G., Mayaux,J., Sacerdot,C., Fromant,M., Springer,M., Grunberg-Manago,M. and Blanquet,S. TITLE Escherichia coli phenylalanyl-tRNA synthetase operon region. Evidence for an attenuation mechanism. Identification of the gene for the ribosomal protein L20 JOURNAL J. Mol. Biol. 171, 239-261 (1983) MEDLINE 84090239 REFERENCE 3 (bases 1313 to 1474; 1932 to 4100) AUTHORS Yanisch-Perron,C., Vieira,J. and Messing,J. TITLE Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors JOURNAL Gene 33, 103-119 (1985) MEDLINE 85180545 REFERENCE 4 (bases 1932 to 3868) AUTHORS Balbas,P., Soberon,X., Merino,E., Zurita,M., Lomeli,H., Valle,F., Flores,N. and Bolivar,F. TITLE Plasmid vector pBR322 and its special-purpose derivatives - A review JOURNAL Gene 50, 3-40 (1986) MEDLINE 87219889 REFERENCE 5 (bases 1476 to 1931) AUTHORS Short,J.M., Fernandez,J.M., Sorge,J.A. and Huse,W.D. TITLE Lambda ZAP: A bacteriophage lambda expression vector with in vivo excision properties JOURNAL Nucleic Acids Res. 16, 7583-7600 (1988) MEDLINE 88319944 REFERENCE 6 (bases 365 to 366; 1026 to 1026) AUTHORS Kast,P. and Hennecke,H. TITLE Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations JOURNAL J. Mol. Biol. 222, 99-124 (1991) MEDLINE 92046090 REFERENCE 7 (bases 3220 to 3255) AUTHORS Lin-Chao,S., Chen,W.-T. and Wong,T.-T. TITLE High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II JOURNAL Mol. Microbiol. 6, 3385-3393 (1992) MEDLINE 93133118 REFERENCE 8 (bases 1 to 55; 1316 to 4099) AUTHORS Alting-Mees,M.A., Sorge,J.A. and Short,J.M. TITLE pBluescriptII: Multifunctional cloning and mapping vectors JOURNAL Meth. Enzymol. 216, 483-495 (1992) MEDLINE 93125166 REFERENCE 9 (bases 4091 to 4133; 1 to 61; 1237 to 1336) AUTHORS Kast,P. TITLE pKSS - A second-generation general purpose cloning vector for efficient positive selection of recombinant clones JOURNAL Gene 138, 109-114 (1994) MEDLINE 94171019 REFERENCE 10 (bases 1 to 4133) AUTHORS Kast,P. TITLE Direct Submission JOURNAL Submitted (10-SEP-1993) Dept. of Chemistry and Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA COMMENT On Oct 6, 1993 this sequence version replaced gi:403935. FEATURES Location/Qualifiers source 1..4133 /organism="unidentified cloning vector" /db_xref="taxon:45196" /lab_host="Escherichia coli" misc_feature 1..61 /note="multiple cloning site I: KpnI-EcoO109I-ApaI-AvaI-XhoI-SalI-AccI-HincII-ClaI-H indIII-EcoRV-EcoRI-AvaI-SmaI" /citation=[9] misc_feature join(1..55,1240..4133) /note="derived from GenBank accession X52331" gene 146..1129 /gene="pheS" /note="Gly294 mutant" CDS 146..1129 /gene="pheS (Gly294 mutant)" /EC_number="6.1.1.20" /function="confers p-Cl-phenylalanine sensitivity" /citation=[2] /codon_start=1 /transl_table=11 /product="phenylalanyl-tRNA synthetase alpha subunit (Gly294 variant)" /protein_id="AAB61946.1" /db_xref="GI:403936" /translation="MSHLAELVASAKAAISQASDVAALDNVRVEYLGKKGHLTLQMTT LRELPPEERPAAGAVINEAKEQVQQALNARKAELESAALNARLAAETIDVSLPGRRIE NGGLHPVTRTIDRIESFFGELGFTVATGPEIEDDYHNFDALNIPGHHPARADHDTFWF DTTRLLRTQTSGVQIRTMKAQQPPIRIIAPGRVYRNDYDQTHTPMFHQMEGLIVDTNI SFTNLKGTLHDFLRNFFEEDLQIRFRPSYFPFTEPSAEVDVMGKNGKWLEVLGCGMVH PNVLRNVGIDPEVYSGFGFGMGMERLTMLRYGVTDLRSFFENDLRFLKQFK" old_sequence 365..366 /gene="pheS" /citation=[2] /replace="GC" gene 1026 /gene="pheS (wild type)" variation 1026 /gene="pheS (wild type)" /note="the exchange of codon GCC (Ala294, wild type) with GGC (Gly294, mutant pheS, as present on pKSS) yielded the phenotypic change from p-Cl-phenylalanine resistance to sensitivity" /citation=[6] /replace="C" misc_feature 1243..1284 /note="multiple cloning site II: BamHI-SpeI-XbaI-EagI-NotI-DsaI-SacII(KspI)-SacI(SstI )" /citation=[9] promoter complement(1292..1311) /standard_name="T7 promoter" /citation=[5] gene 1314..>1474 /gene="'lacZ'" CDS 1314..>1474 /gene="lacZ" /EC_number="3.2.1.23" /note="N-truncated alpha-fragment of beta-galactosidase; blue/white screening with the lacZ system is irrelevant in pKSS" /citation=[5] /codon_start=3 /transl_table=11 /product="beta-galactosidase fragment" /protein_id="AAB61947.1" /db_xref="GI:403937" /translation="SLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQ QLRSLNGEW" primer_bind complement(1318..1334) /note="annealing site for universal M13(-20) sequencing primer" /citation=[5] rep_origin 1476..1931 /standard_name="f1 phage origin region for single strand DNA replication" /note="nicking site of gene II protein is between pos. 1768 and 1769" /citation=[5] /direction=right old_sequence 1922 /citation=[5] /replace="T" gene 2062..2922 /gene="bla" CDS 2062..2922 /gene="bla" /EC_number="3.5.2.6" /function="provides resistance to ampicillin" /citation=[3] /codon_start=1 /transl_table=11 /product="pre-beta-lactamase" /protein_id="AAB61948.1" /db_xref="GI:403938" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" sig_peptide 2062..2130 /gene="bla" mat_peptide 2131..2919 /gene="bla" /EC_number="3.5.2.6" /product="beta-lactamase" variation 2305 /gene="bla" /note="mutation of HincII site of pBR322 [3]" /citation=[3] /replace="G" variation 2606 /gene="bla" /note="mutation of PstI site of pBR322 [3]" /citation=[3] /replace="C" rep_origin 3082..3696 /standard_name="pMB1 (ColE1-like) plasmid origin of replication, as in pBR322 [4]" /note="the DNA region indicated is sufficient for a functional replication origin [1]; DNA replication initiates at pos. 3681 to 3683 [4]" /citation=[1] /direction=right old_sequence 3240 /note="according to [7], the T at position 3240 is responsible for the higher copy number of pUC derivatives [3,8], including pKSS, compared to their parent pBR322 [1,4]" /citation=[1] /replace="C" gene <3864..3956 /gene="'lacI" CDS <3864..3956 /gene="'lacI" /note="vestigal C-terminal portion of lac repressor; this 3' portion of lacI is not functional" /citation=[3] /codon_start=1 /transl_table=11 /product="lac repressor fragment" /protein_id="AAB61949.1" /db_xref="GI:403939" /translation="APNTQTASPRALADSLMQLARQVSRLESGQ" protein_bind 3957..3994 /standard_name="CAP binding site (wild-type sequence)" /citation=[5] /bound_moiety="catabolite activator protein (CAP)" promoter 3957..4061 /standard_name="lac promoter-operator region (wild type)" /note="for details and references to the lac promoter-operator region see [(in) Miller,J.H. and Reznikoff,W.S. (eds); The Operon; 2nd ed (1980); Cold Spring Harbor; pp.177-243.]" /citation=[5] -35_signal 4005..4010 /gene="lacZ'" gene 4005..4093 /gene="lacZ'" -10_signal 4029..4034 /gene="lacZ'" protein_bind 4041..4061 /gene="lacZ'" /standard_name="lacO; lac operator" /bound_moiety="lac repressor protein" CDS 4079..>4093 /gene="lacZ'" /note="N-terminal 5 residues of beta-galactosidase" /citation=[5] /codon_start=1 /transl_table=11 /product="beta-galactosidase fragment" /protein_id="AAB61950.1" /db_xref="GI:2213571" /translation="MTMIT" promoter 4104..4123 /standard_name="T3 promoter" /citation=[5] old_sequence 4133 /note="all pBluescript I KS (+/-) vectors (and pKSS) exhibit the deletion of the G nucleotide 5' to the KpnI site [8,9]" /citation=[5] /replace="TG" BASE COUNT 1010 a 1052 c 1053 g 1018 t ORIGIN 1 ggtaccgggc cccccctcga ggtcgacggt atcgataagc ttgatatcga attcccccgg 61 gaccaaaatg gcaagtaaaa tagcctgatg ggataggctc taagtccaac gaaccagtgt 121 caccactgac acaatgagga aaaccatgtc acatctcgca gaactggttg ccagtgcgaa 181 ggcggccatt agccaggcgt cagatgttgc cgcgttagat aatgtgcgcg tcgaatattt 241 gggtaaaaaa gggcacttaa cccttcagat gacgaccctg cgtgagctgc cgccagaaga 301 gcgtccggca gctggtgcgg ttatcaacga agcgaaagag caggttcagc aggcgctgaa 361 tgcgcgtaaa gcggaactgg aaagcgctgc actgaatgcg cgtctggcgg cggaaacgat 421 tgatgtctct ctgccaggtc gtcgcattga aaacggcggt ctgcatccgg ttacccgtac 481 catcgaccgt atcgaaagtt tcttcggtga gcttggcttt accgtggcaa ccgggccgga 541 aatcgaagac gattatcata acttcgatgc tctgaacatt cctggtcacc acccggcgcg 601 cgctgaccac gacactttct ggtttgacac tacccgcctg ctgcgtaccc agacctctgg 661 cgtacagatc cgcaccatga aagcccagca gccaccgatt cgtatcatcg cgcctggccg 721 tgtttatcgt aacgactacg accagactca cacgccgatg ttccatcaga tggaaggtct 781 gattgttgat accaacatca gctttaccaa cctgaaaggc acgctgcacg acttcctgcg 841 taacttcttt gaggaagatt tgcagattcg cttccgtcct tcctacttcc cgtttaccga 901 accttctgca gaagtggacg tcatgggtaa aaacggtaaa tggctggaag tgctgggctg 961 cgggatggtg catccgaacg tgttgcgtaa cgttggcatc gacccggaag tttactctgg 1021 tttcggcttc gggatgggga tggagcgtct gactatgttg cgttacggcg tcaccgacct 1081 gcgttcattc ttcgaaaacg atctgcgttt cctcaaacag tttaaataag gcaggaatag 1141 attatgaaat tcagtgaact gtggttacgc gaatgggtga acccggcgat tgatagcgat 1201 gcgctggcaa atcaaatcac tatggcgggc ctggaagttg ggggatccac tagttctaga 1261 gcggccgcca ccgcggtgga gctccaattc gccctatagt gagtcgtatt acaattcact 1321 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 1381 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 1441 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag 1501 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 1561 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 1621 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 1681 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 1741 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 1801 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 1861 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 1921 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 1981 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 2041 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 2101 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 2161 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 2221 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 2281 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 2341 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 2401 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 2461 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 2521 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 2581 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 2641 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 2701 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 2761 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 2821 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 2881 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 2941 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 3001 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 3061 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 3121 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 3181 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt 3241 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 3301 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 3361 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 3421 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 3481 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 3541 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 3601 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 3661 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 3721 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 3781 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 3841 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 3901 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc 3961 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt 4021 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat 4081 gaccatgatt acgccaagct cgaaattaac cctcactaaa gggaacaaaa gct //
Primers	aacagctatgaccatg reverse primer, attaaccctcactaaag T3 primer, cgaggtcgacggtatcg KS primer, tctagaactagtggatc SK primer, aatacgactcactatag T7 primer, gtaaaacgacggccagt M13 primer, gttttcccagtcacgac -40 primer

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