LOCUS pCYB3 6846 bp DNA circular 10-DEC-2007 DEFINITION Cloning vector pCYB3, complete sequence. ACCESSION VERSION KEYWORDS . SOURCE Cloning vector pCYB3 ORGANISM Cloning vector pCYB3 other sequences; artificial sequences; vectors. REFERENCE 1 (bases 1 to 6846) 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 6846) AUTHORS Perler,F.B. TITLE InBase, the intein database JOURNAL Nucleic Acids Res 30 (1), 383-384 (2002) PUBMED 11752343 REFERENCE 3 (bases 1 to 6846) AUTHORS Ghosh,I. and Xu,M.Q. TITLE Direct Submission JOURNAL Submitted (10-DEC-2007) Research Department, New England Biolabs, 240 County Road, Ipswich, MA 01938, USA FEATURES Location/Qualifiers source 1..6846 /organism="Cloning vector pCYB3" /mol_type="other DNA" gene 15..1163 /gene="lacIq" promoter 15..46 /gene="lacIq" /note="lacIq promoter (overexpressing mutant) (clockwise)" CDS 81..1163 /gene="lacIq" /exception="alternative start codon" /codon_start=1 /transl_table=11 /product="lac repressor (LacI)" /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT QTASPRALADSLMQLARQVSRLESGQ" promoter 1406..1433 /note="Ptac promoter (clockwise)" protein_bind 1438..1462 /note="lac operator" /bound_moiety="LacI" primer_bind 1446..1469 /note="Ptac forward sequencing primer (NEB #1260, discontinued)" RBS 1515..1518 /note="RBS for expression CDS" misc_feature 1526..1575 /gene="expression CDS" /note="multiple cloning site (NcoI-SapI)" gene 1528..3129 /gene="expression CDS" CDS 1528..3129 /gene="expression CDS" /note="multiple cloning site (NcoI-SapI) 1526..1575; Sce VMA intein sequence 1576..2937; chitin binding domain (CBD) sequence 2971..3129" /codon_start=1 /product="intein/CBD fusion" /translation="MASSRVDSREFLEGSSCFAKGTNVLMADGSIECIENIEVGNKVM GKDGRPREVIKLPRGRETMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTP RSVRRLSRTIKGVEYFEVITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVE SYRKASNKAYFEWTIEARDLSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTI EGPKVLAYLLGLWIGDGLSDRATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQV AKTVNLYSKVVRGNGIRNNLNTENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRETF LAGLIDSDGYVTDEHGIKATIKTIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVTK HKISYAIYMSGGDVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKEDDYYGI TLSDDSDHQFLLGSQVVVHACGGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGKT YKCLQPHTSLAGWEPSNVPALWQLQ" primer_bind complement(1639..1662) /note="intein reverse primer sequence (NEB #1261S)" gene 3677..4537 /gene="bla" CDS 3677..4537 /gene="bla" /note="ampR (confers resistance to ampicillin)" /codon_start=1 /product="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" sig_peptide 3677..3745 /gene="bla" /note="required for secretion to the periplasm; cleaved off to form the mature beta-lactamase protein" rep_origin 4579..5092 /note="M13 origin of replication (+ -)" rep_origin 5203..5791 /note="pMB1 origin of replication (clockwise) (RNAII -35 to RNA/DNA switch point)" gene complement(6160..6351) /gene="rop" CDS complement(6160..6351) /gene="rop" /exception="alternative start codon" /codon_start=1 /transl_table=11 /product="ROP protein" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH ADELYRSCLARFGDDGENL" BASE COUNT 1736 a 1623 c 1779 g 1708 t ORIGIN 1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga 61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg 121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa 181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac 241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc 301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg 361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc 421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca 481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga 541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc 601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg 661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag 721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga 781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa 841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg 901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc 961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata 1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg 1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga 1501 gcacttcacc aacaaggacc atagaccatg gctagctcgc gagtcgactc tagagaattc 1561 ctcgagggct cttcctgctt tgccaagggt accaatgttt taatggcgga tgggtctatt 1621 gaatgtattg aaaacattga ggttggtaat aaggtcatgg gtaaagatgg cagacctcgt 1681 gaggtaatta aattgcccag aggaagagaa actatgtaca gcgtcgtgca gaaaagtcag 1741 cacagagccc acaaaagtga ctcaagtcgt gaagtgccag aattactcaa gtttacgtgt 1801 aatgcgaccc atgagttggt tgttagaaca cctcgtagtg tccgccgttt gtctcgtacc 1861 attaagggtg tcgaatattt tgaagttatt acttttgaga tgggccaaaa gaaagccccc 1921 gacggtagaa ttgttgagct tgtcaaggaa gtttcaaaga gctacccaat atctgagggg 1981 cctgagagag ccaacgaatt agtagaatcc tatagaaagg cttcaaataa agcttatttt 2041 gagtggacta ttgaggccag agatctttct ctgttgggtt cccatgttcg taaagctacc 2101 taccagactt acgctccaat tctttatgag aatgaccact ttttcgacta catgcaaaaa 2161 agtaagtttc atctcaccat tgaaggtcca aaagtacttg cttatttact tggtttatgg 2221 attggtgatg gattgtctga cagggcaact ttttcggttg attccagaga tacttctttg 2281 atggaacgtg ttactgaata tgctgaaaag ttgaatttgt gcgccgagta taaggacaga 2341 aaagaaccac aagttgccaa aactgttaat ttgtactcta aagttgtcag aggtaatggt 2401 attcgcaata atcttaatac tgagaatcca ttatgggacg ctattgttgg cttaggattc 2461 ttgaaggacg gtgtcaaaaa tattccttct ttcttgtcta cggacaatat cggtactcgt 2521 gaaacatttc ttgctggtct aattgattct gatggctatg ttactgatga gcatggtatt 2581 aaagcaacaa taaagacaat tcatacttct gtcagagatg gtttggtttc ccttgctcgt 2641 tctttaggct tagtagtctc ggttaacgca gaacctgcta aggttgacat gaatgtcacc 2701 aaacataaaa ttagttatgc tatttatatg tctggtggag atgttttgct taacgttctt 2761 tcgaagtgtg ccggctctaa aaaattcagg cctgctcccg ccgctgcttt tgcacgtgag 2821 tgccgcggat tttatttcga gttacaagaa ttgaaggaag acgattatta tgggattact 2881 ttatctgatg attctgatca tcagtttttg cttggatccc aggttgtcgt ccatgcatgc 2941 ggtggcctga ccggtctgaa ctcaggcctc acgacaaatc ctggtgtatc cgcttggcag 3001 gtcaacacag cttatactgc gggacaattg gtcacatata acggcaagac gtataaatgt 3061 ttgcagcccc acacctcctt ggcaggatgg gaaccatcca acgttcctgc cttgtggcag 3121 cttcaatgac tgcagcctga atggcgaatg gcagcttggc tgttttggcg gatgagataa 3181 gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt 3241 gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg 3301 ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc 3361 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg 3421 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac 3481 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga 3541 aggccatcct gacggatggc ctttttgcgt ttctacaaac tctttttgtt tatttttcta 3601 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 3661 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 3721 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 3781 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 3841 tgagagtttt cgccccgaag aacgttctcc aatgatgagc acttttaaag ttctgctatg 3901 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 3961 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 4021 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 4081 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 4141 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 4201 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgtaaactat taactggcga 4261 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 4321 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 4381 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 4441 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 4501 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 4561 tatactttag attgatttac cccggttgat aatcagaaaa gccccaaaaa caggaagatt 4621 gtataagcaa atatttaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt 4681 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca 4741 aaagaatagc ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta 4801 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta 4861 cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg 4921 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga 4981 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg 5041 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtaaaaggat 5101 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 5161 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 5221 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 5281 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 5341 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 5401 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 5461 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 5521 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 5581 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 5641 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 5701 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 5761 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 5821 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 5881 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 5941 gcgcagcgag tcagtgagcg aggaagctat ggtgcactct cagtacaatc tgctctgatg 6001 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc 6061 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 6121 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 6181 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgca gcgattcaca 6241 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg 6301 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca cttgatgcct 6361 ccgtgtaagg gggaatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg 6421 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa 6481 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc 6541 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc 6601 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa 6661 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac 6721 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc 6781 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc aacgctgccc 6841 gaaatt //