LOCUS pCYB4 6843 bp DNA circular 10-DEC-2007 DEFINITION Cloning vector pCYB4, complete sequence. ACCESSION VERSION KEYWORDS . SOURCE Cloning vector pCYB4 ORGANISM Cloning vector pCYB4 other sequences; artificial sequences; vectors. REFERENCE 1 (bases 1 to 6843) 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 6843) 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 6843) 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..6843 /organism="Cloning vector pCYB4" /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..1572 /gene="expression CDS" /note="multiple cloning site (NcoI-SmaI)" gene 1528..3126 /gene="expression CDS" CDS 1528..3126 /gene="expression CDS" /note="multiple cloning site (NcoI-SmaI) 1526..1572; Sce VMA intein sequence 1573..2934; chitin binding domain (CBD) sequence 2968..3126" /codon_start=1 /product="intein/CBD fusion" /translation="MASSRVDSREFLEPGCFAKGTNVLMADGSIECIENIEVGNKVMG KDGRPREVIKLPRGRETMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPR SVRRLSRTIKGVEYFEVITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVES YRKASNKAYFEWTIEARDLSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIE GPKVLAYLLGLWIGDGLSDRATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVA KTVNLYSKVVRGNGIRNNLNTENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRETFL AGLIDSDGYVTDEHGIKATIKTIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVTKH KISYAIYMSGGDVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKEDDYYGIT LSDDSDHQFLLGSQVVVHACGGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTY KCLQPHTSLAGWEPSNVPALWQLQ" primer_bind complement(1636..1659) /note="intein reverse primer sequence (NEB #1261S)" gene 3674..4534 /gene="bla" CDS 3674..4534 /gene="bla" /note="ampR (confers resistance to ampicillin)" /codon_start=1 /product="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" sig_peptide 3674..3742 /gene="bla" /note="required for secretion to the periplasm; cleaved off to form the mature beta-lactamase protein" rep_origin 4576..5089 /note="M13 origin of replication (+ -)" rep_origin 5200..5788 /note="pMB1 origin of replication (clockwise) (RNAII -35 to RNA/DNA switch point)" gene complement(6157..6348) /gene="rop" CDS complement(6157..6348) /gene="rop" /exception="alternative start codon" /codon_start=1 /transl_table=11 /product="ROP protein" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH ADELYRSCLARFGDDGENL" BASE COUNT 1736 a 1622 c 1780 g 1705 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 ctcgagcccg ggtgctttgc caagggtacc aatgttttaa tggcggatgg gtctattgaa 1621 tgtattgaaa acattgaggt tggtaataag gtcatgggta aagatggcag acctcgtgag 1681 gtaattaaat tgcccagagg aagagaaact atgtacagcg tcgtgcagaa aagtcagcac 1741 agagcccaca aaagtgactc aagtcgtgaa gtgccagaat tactcaagtt tacgtgtaat 1801 gcgacccatg agttggttgt tagaacacct cgtagtgtcc gccgtttgtc tcgtaccatt 1861 aagggtgtcg aatattttga agttattact tttgagatgg gccaaaagaa agcccccgac 1921 ggtagaattg ttgagcttgt caaggaagtt tcaaagagct acccaatatc tgaggggcct 1981 gagagagcca acgaattagt agaatcctat agaaaggctt caaataaagc ttattttgag 2041 tggactattg aggccagaga tctttctctg ttgggttccc atgttcgtaa agctacctac 2101 cagacttacg ctccaattct ttatgagaat gaccactttt tcgactacat gcaaaaaagt 2161 aagtttcatc tcaccattga aggtccaaaa gtacttgctt atttacttgg tttatggatt 2221 ggtgatggat tgtctgacag ggcaactttt tcggttgatt ccagagatac ttctttgatg 2281 gaacgtgtta ctgaatatgc tgaaaagttg aatttgtgcg ccgagtataa ggacagaaaa 2341 gaaccacaag ttgccaaaac tgttaatttg tactctaaag ttgtcagagg taatggtatt 2401 cgcaataatc ttaatactga gaatccatta tgggacgcta ttgttggctt aggattcttg 2461 aaggacggtg tcaaaaatat tccttctttc ttgtctacgg acaatatcgg tactcgtgaa 2521 acatttcttg ctggtctaat tgattctgat ggctatgtta ctgatgagca tggtattaaa 2581 gcaacaataa agacaattca tacttctgtc agagatggtt tggtttccct tgctcgttct 2641 ttaggcttag tagtctcggt taacgcagaa cctgctaagg ttgacatgaa tgtcaccaaa 2701 cataaaatta gttatgctat ttatatgtct ggtggagatg ttttgcttaa cgttctttcg 2761 aagtgtgccg gctctaaaaa attcaggcct gctcccgccg ctgcttttgc acgtgagtgc 2821 cgcggatttt atttcgagtt acaagaattg aaggaagacg attattatgg gattacttta 2881 tctgatgatt ctgatcatca gtttttgctt ggatcccagg ttgtcgtcca tgcatgcggt 2941 ggcctgaccg gtctgaactc aggcctcacg acaaatcctg gtgtatccgc ttggcaggtc 3001 aacacagctt atactgcggg acaattggtc acatataacg gcaagacgta taaatgtttg 3061 cagccccaca cctccttggc aggatgggaa ccatccaacg ttcctgcctt gtggcagctt 3121 caatgactgc agcctgaatg gcgaatggca gcttggctgt tttggcggat gagataagat 3181 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 3241 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 3301 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 3361 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 3421 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 3481 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 3541 ccatcctgac ggatggcctt tttgcgtttc tacaaactct ttttgtttat ttttctaaat 3601 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 3661 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 3721 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 3781 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 3841 gagttttcgc cccgaagaac gttctccaat gatgagcact tttaaagttc tgctatgtgg 3901 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc 3961 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 4021 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 4081 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 4141 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 4201 tgacaccacg atgcctgtag caatggcaac aacgttgcgt aaactattaa ctggcgaact 4261 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 4321 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 4381 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 4441 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 4501 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 4561 actttagatt gatttacccc ggttgataat cagaaaagcc ccaaaaacag gaagattgta 4621 taagcaaata tttaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt 4681 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 4741 gaatagcccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 4801 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 4861 gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 4921 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 4981 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 5041 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgta aaaggatcta 5101 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 5161 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5221 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 5281 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 5341 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 5401 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 5461 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 5521 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 5581 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 5641 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 5701 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 5761 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 5821 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 5881 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 5941 cagcgagtca gtgagcgagg aagctatggt gcactctcag tacaatctgc tctgatgccg 6001 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg 6061 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 6121 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 6181 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgcagcg attcacagat 6241 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct 6301 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactt gatgcctccg 6361 tgtaaggggg aatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc 6421 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa 6481 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc 6541 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg 6601 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg 6661 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt 6721 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg 6781 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgaa 6841 att //