LOCUS pKYB1 8393 bp DNA circular 20-NOV-2007 DEFINITION Cloning vector pKYB1, complete sequence. ACCESSION VERSION KEYWORDS . SOURCE Cloning vector pKYB1 ORGANISM Cloning vector pKYB1 Unclassified. REFERENCE 1 (bases 1 to 8393) AUTHORS Chong,S., Mersha,F.B., Comb,D.G., Scott,M.E., Landry,D., Vence,L.M., Perler,F.B., Benner,J., Kucera,R.B., Hirvonen,C.A., Pelletier,J.J., Paulus,H. and Xu,M.-Q. TITLE Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element JOURNAL Gene 192, 271-281 (1997) PUBMED 9224900 REFERENCE 2 (bases 1 to 8393) AUTHORS Perler,F.B. TITLE InBase, the intein database JOURNAL Nucleic Acids Res 30 (1), 383-384 (?) PUBMED 11752343 REFERENCE 3 (bases 1 to 8393) AUTHORS Ghosh,I. and Xu,M.Q. TITLE Direct Submission JOURNAL Submitted (20-NOV-2007) Research Department, New England Biolabs, 240 County Road, Ipswich, MA 01938, USA COMMENT Plasmid renumbered relative to older pKYB1 sequence files, shifted by 7: new nt 1 was old nt 8. FEATURES Location/Qualifiers source 1..8393 /organism="Cloning vector pKYB1" /mol_type="other DNA" gene 415..1230 /gene="aph(3')-Ia" CDS 415..1230 /gene="aph(3')-Ia" /note="kanR (confers resistance to kanamycin)" /codon_start=1 /product="aminoglycoside phosphotransferase from Tn903" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" rep_origin 1958..2471 /note="M13 origin of replication (+ -)" rep_origin 2582..3170 /note="pMB1 origin of replication (clockwise) (RNAII -35 to RNA/DNA switch point)" gene complement(3539..3730) /gene="rop" CDS complement(3539..3730) /gene="rop" /exception="alternative start codon" /codon_start=1 /transl_table=11 /product="ROP protein" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH ADELYRSCLARFGDDGENL" gene complement(4287..5369) /gene="lacI" CDS complement(4287..5369) /gene="lacI" /exception="alternative start codon" /codon_start=1 /transl_table=11 /product="lac repressor (LacI)" /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT QTASPRALADSLMQLARQVSRLESGQ" primer_bind 6553..6572 /note="T7 universal primer sequence (NEB #S1248S)" promoter 6553..6570 /note="T7 promoter (transcript start 6570 clockwise)" protein_bind 6572..6596 /note="lac operator" /bound_moiety="LacI" RBS 6626..6632 /note="RBS for expression CDS" misc_feature 6638..6691 /gene="expression CDS" /note="multiple cloning site (NdeI-SapI)" gene 6641..8245 /gene="expression CDS" CDS 6641..8245 /gene="expression CDS" /note="multiple cloning site (NdeI-SapI) 6638..6691; Sce VMA intein sequence 6692..8053; chitin binding domain (CBD) sequence 8087..8245" /codon_start=1 /product="intein/CBD fusion" /translation="MASSRVDGGREFLEGSSCFAKGTNVLMADGSIECIENIEVGNKV MGKDGRPREVIKLPRGRETMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRT PRSVRRLSRTIKGVEYFEVITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELV ESYRKASNKAYFEWTIEARDLSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLT IEGPKVLAYLLGLWIGDGLSDRATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQ VAKTVNLYSKVVRGNGIRNNLNTENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRET FLAGLIDSDGYVTDEHGIKATIKTIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVT KHKISYAIYMSGGDVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKEDDYYG ITLSDDSDHQFLLGSQVVVHACGGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGK TYKCLQPHTSLAGWEPSNVPALWQLQ" primer_bind 6755..6778 /note="intein reverse primer sequence (NEB #1261S)" terminator 8265..8387 /note="T7 Tphi transcription terminator" BASE COUNT 2114 a 1998 c 2201 g 2080 t ORIGIN 1 cagccttttt tctcctgcca catgaagcac ttcactgaca ccctcatcag tgccaacata 61 gtaagccagt atacactccg ctagcgctga ggtctgcctc gtgaagaagg tgttgctgac 121 tcataccagg cctgaatcgc cccatcatcc agccagaaag tgagggagcc acggttgatg 181 agagctttgt tgtaggtgga ccagttggtg attttgaact tttgctttgc cacggaacgg 241 tctgcgttgt cgggaagatg cgtgatctga tccttcaact cagcaaaagt tcgatttatt 301 caacaaagcc acgttgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata 361 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc 421 catattcaac gggaaacgtc ttgctcgcgt ccgcgattaa attccaacat ggatgctgat 481 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 541 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 601 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 661 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 721 ggcaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 781 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 841 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 901 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 961 cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 1021 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 1081 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 1141 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 1201 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg 1261 cagagcatta cgctgacttg acgggacggc ggctttgttg agacgccggg caagagcaac 1321 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 1381 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 1441 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 1501 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 1561 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 1621 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 1681 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 1741 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 1801 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 1861 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 1921 cagaccaagt ttactcatat atactttaga ttgatttacc ccggttgata atcagaaaag 1981 ccccaaaaac aggaagattg tataagcaaa tatttaaatt gtaaacgtta atattttgtt 2041 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg 2101 caaaatccct tataaatcaa aagaatagcc cgagataggg ttgagtgttg ttccagtttg 2161 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta 2221 tcagggcgat ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg 2281 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa 2341 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct 2401 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct 2461 acagggcgcg taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 2521 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 2581 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 2641 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 2701 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 2761 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 2821 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 2881 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 2941 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 3001 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 3061 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 3121 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 3181 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 3241 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 3301 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagctatg gtgcactctc 3361 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg 3421 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 3481 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 3541 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt 3601 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct 3661 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct 3721 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga 3781 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg 3841 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc 3901 agggtcaatg ccagccgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg 3961 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag 4021 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc 4081 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca 4141 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg 4201 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga 4261 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 4321 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc 4381 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg 4441 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt 4501 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact 4561 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc 4621 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc 4681 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga 4741 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa 4801 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg 4861 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag 4921 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg 4981 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc 5041 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc 5101 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga 5161 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg 5221 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa 5281 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct 5341 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg 5401 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg 5461 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag 5521 caccgccgcc gcaaggaatg gtgcatgccg gcatgccgcc ctttcgtctt caagaattaa 5581 ttcccaattc cccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg 5641 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga 5701 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc 5761 caggaattaa ttccccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt 5821 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc 5881 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 5941 tgccaggaat taattcccca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 6001 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg 6061 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata 6121 aactgccagg aattaattcc ccaggcatca aataaaacga aaggctcagt cgaaagactg 6181 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc 6241 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc 6301 ataaactgcc aggaattaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga 6361 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc 6421 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc 6481 gccataaact gccaggaatt ggggatcgga attaattccc ggtttaaacc ggggatctcg 6541 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc 6601 tagaaataat tttgtttaac tttaagaagg agatatacat atggctagct cgcgagtcga 6661 cggcggccgc gaattcctcg agggctcttc ctgctttgcc aagggtacca atgttttaat 6721 ggcggatggg tctattgaat gtattgaaaa cattgaggtt ggtaataagg tcatgggtaa 6781 agatggcaga cctcgtgagg taattaaatt gcccagagga agagaaacta tgtacagcgt 6841 cgtgcagaaa agtcagcaca gagcccacaa aagtgactca agtcgtgaag tgccagaatt 6901 actcaagttt acgtgtaatg cgacccatga gttggttgtt agaacacctc gtagtgtccg 6961 ccgtttgtct cgtaccatta agggtgtcga atattttgaa gttattactt ttgagatggg 7021 ccaaaagaaa gcccccgacg gtagaattgt tgagcttgtc aaggaagttt caaagagcta 7081 cccaatatct gaggggcctg agagagccaa cgaattagta gaatcctata gaaaggcttc 7141 aaataaagct tattttgagt ggactattga ggccagagat ctttctctgt tgggttccca 7201 tgttcgtaaa gctacctacc agacttacgc tccaattctt tatgagaatg accacttttt 7261 cgactacatg caaaaaagta agtttcatct caccattgaa ggtccaaaag tacttgctta 7321 tttacttggt ttatggattg gtgatggatt gtctgacagg gcaacttttt cggttgattc 7381 cagagatact tctttgatgg aacgtgttac tgaatatgct gaaaagttga atttgtgcgc 7441 cgagtataag gacagaaaag aaccacaagt tgccaaaact gttaatttgt actctaaagt 7501 tgtcagaggt aatggtattc gcaataatct taatactgag aatccattat gggacgctat 7561 tgttggctta ggattcttga aggacggtgt caaaaatatt ccttctttct tgtctacgga 7621 caatatcggt actcgtgaaa catttcttgc tggtctaatt gattctgatg gctatgttac 7681 tgatgagcat ggtattaaag caacaataaa gacaattcat acttctgtca gagatggttt 7741 ggtttccctt gctcgttctt taggcttagt agtctcggtt aacgcagaac ctgctaaggt 7801 tgacatgaat gtcaccaaac ataaaattag ttatgctatt tatatgtctg gtggagatgt 7861 tttgcttaac gttctttcga agtgtgccgg ctctaaaaaa ttcaggcctg ctcccgccgc 7921 tgcttttgca cgtgagtgcc gcggatttta tttcgagtta caagaattga aggaagacga 7981 ttattatggg attactttat ctgatgattc tgatcatcag tttttgcttg gatcccaggt 8041 tgtcgtccat gcatgcggtg gcctgaccgg tctgaactca ggcctcacga caaatcctgg 8101 tgtatccgct tggcaggtca acacagctta tactgcggga caattggtca catataacgg 8161 caagacgtat aaatgtttgc agccccacac ctccttggca ggatgggaac catccaacgt 8221 tcctgccttg tggcagcttc aatgactgca ggaaggggat ccggctgcta acaaagcccg 8281 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 8341 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gtg //