LOCUS pACBSR 7347 bp DNA circular SYN 17-JUL-2001 DEFINITION Mutagenesis plasmid pACBSR, complete sequence (not verified). ACCESSION . VERSION . KEYWORDS . SOURCE Mutagenesis plasmid pACBSR ORGANISM Mutagenesis plasmid pACBSR other sequences; artificial sequences; vectors. REFERENCE 1 (description) AUTHORS Herring, C.D., Glasner, J.D., and Blattner,F.R. TITLE Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. JOURNAL Gene 311, 153-163 (2003) PUBMED 12853150 COMMENT Theoretical sequence, constructed from sequences of pBAD18-Kan (ATCC 87397; J. Beckwith), lambda (GenBank: J02459.1), pSCM525 (B. Dujon), and pACYC184 (GenBank: X06403.1). Plasmid constuction: pBAD18-Kan was digested with NheI and PstI, then the Nhe/Pst fragment of the I-SceI gene OF131/132 PCR product was ligated in. This gave "pBAD/Sce". It was then digested with SphI and the SphI-digested lambda Red OF231/233 PCR product was ligated in, giving "pBSR." Then the 5.3Kb gel purified Alw44I fragement of pBSR was ligated to the Alw44I- digested pACYC184 Cam/ori OF253/254 PCR product, giving "pACBSR". NOTE: The orientation of the Cam/ori fragment relative to the other segment was not determined, so this sequence may be in error in that regard. FEATURES Location/Qualifiers CDS complement(533..1411) /gene="araC" /codon_start=1 /transl_table=11 /product="arabinose operon regulatory protein" /db_xref="UniProtKB/Swiss-Prot:P0A9E0" /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKG YILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRA YWHEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLL LRRMEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQ LGISVLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEF RAGCEEKVNDVAVKLS" misc_feature 1440..1457 /note="operator O2" promoter complement(1562..1590) /note="araC promoter" misc_feature 1598..1619 /note="operator O1" misc_feature 1641..1654 /note="CAP site" misc_feature 1650..1688 /note="operator I2 + I1" promoter 1687..1714 /note="araBAD promoter" misc_feature 1737..4667 /note="multiple cloning site" misc_feature 1743..2481 /note="Sce fragment: OF131/132 PCR product NheI/PstI restriction fragment" CDS 1757..2470 /gene="I.SceI" /codon_start=1 /transl_table=11 /product="intron-encoded endonuclease I-SceI" /EC_number="3.1.-.-" /db_xref="PDB:3OOL_A" /translation="MHMKNIKKNQVMNLGPNSKLLKEYKSQLIELNIEQFEAGIGLIL GDAYIRSRDEGKTYCMQFEWKNKAYMDHVCLLYDQWVLSPPHKKERVNHLGNLVITWG AQTFKHQAFNKLANLFIVNNKKTIPNNLVENYLTPMSLAYWFMDDGGKWDYNKNSTNK SIVLNTQSFTFEEVEYLVKGLRNKFQLNCYVKINKNKPIIYIDSMSYLIFYNLIKPYL IPQMMYKLPNTISSETFLK" RBS 2494..2501 /note="RBS - optimized/synthetic" misc_feature 2494..4655 /note="Red fragment: SphI digested OF231/233 PCR product" CDS 2511..2927 /gene="gam" /codon_start=1 /transl_table=11 /product="host-nuclease inhibitor protein gam" /db_xref="UniProtKB/Swiss-Prot:P03702" /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNA YYIQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKS ITRAFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV" CDS 2933..3718 /gene="bet" /codon_start=1 /transl_table=11 /product="recombination protein bet" /db_xref="UniProtKB/Swiss-Prot:P03698" /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQF IALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNE SCTCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCA RLAFGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDD DLLPLCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA" CDS 3715..4395 /gene="exo" /codon_start=1 /transl_table=11 /product="exonuclease" /EC_number="3.1.11.3" /db_xref="UniProtKB/Swiss-Prot:P03697" /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKP RSGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTE SPIIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQY SMWVTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIG FVFGEQWR" misc_recomb complement(4476^4477) /note="lambda::lambdoid hybridization site" terminator 4668..5093 /note="rrnB T1 T2" promoter 5148..5154 /note="bla P3 promoter" misc_feature 5307..7347 /note="pACYC184-derived fragment: Alw44I digested OF253/254 PCR product" /note="this fragement may be in the reverse orientation" CDS complement(5386..6045) /gene="cat" /codon_start=1 /transl_table=11 /product="chloramphenicol acetyltransferase" /EC_number="2.3.1.28" /note="chloramphenicol resistance" /db_xref="UniProtKB/Swiss-Prot:Q7AK95" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG A" rep_origin 6407..7319 /note="p15A origin of replication" BASE COUNT 1951 a 1864 c 1772 g 1760 t ORIGIN 1 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta 61 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc 121 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 181 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagca aggagatggc 241 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 301 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 361 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatctgctc 421 atgtttgaca gcttatcatc gatgcataat gtgcctgtca aatggacgaa gcagggattc 481 tgcaaaccct atgctactcc gtcaagccgt caattgtctg attcgttacc aattatgaca 541 acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg 601 ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc 661 gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac 721 gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca 781 gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg 841 ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg attatccatc ggtggatgga 901 gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga tttatcgcca 961 gcagctccga atagcgccct tccccttgcc cggcgttaat gatttgccca aacaggtcgc 1021 tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc cgtattggca aatattgacg 1081 gccagttaag ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc 1141 attcgcgagc ctccggatga cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa 1201 tatcacccgg tcggcaaaca aattctcgtc cctgattttt caccaccccc tgaccgcgaa 1261 tggtgagatt gagaatataa cctttcattc ccagcggtcg gtcgataaaa aaatcgagat 1321 aaccgttggc ctcaatcggc gttaaacccg ccaccagatg ggcattaaac gagtatcccg 1381 gcagcagggg atcattttgc gcttcagcca tacttttcat actcccgcca ttcagagaag 1441 aaaccaattg tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct 1501 cgctaaccaa accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag 1561 ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt 1621 atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc 1681 ctacctgacg ctttttatcg caactctcta ctgtttctcc atacccgttt ttttgggcta 1741 gcaggagggt acctatatgc atatgaaaaa catcaaaaaa aaccaggtaa tgaacctggg 1801 tccgaactct aaactgctga aagaatacaa atcccagctg atcgaactga acatcgaaca 1861 gttcgaagca ggtatcggtc tgatcctggg tgatgcttac atccgttctc gtgatgaagg 1921 taaaacctac tgtatgcagt tcgagtggaa aaacaaagca tacatggacc acgtatgtct 1981 gctgtacgat cagtgggtac tgtccccgcc gcacaaaaaa gaacgtgtta accacctggg 2041 taacctggta atcacctggg gcgcccagac tttcaaacac caagctttca acaaactggc 2101 taacctgttc atcgttaaca acaaaaaaac catcccgaac aacctggttg aaaactacct 2161 gaccccgatg tctctggcat actggttcat ggatgatggt ggtaaatggg attacaacaa 2221 aaactctacc aacaaatcga tcgtactgaa cacccagtct ttcactttcg aagaagtaga 2281 atacctggtt aagggtctgc gtaacaaatt ccaactgaac tgttacgtaa aaatcaacaa 2341 aaacaaaccg atcatctaca tcgattctat gtcttacctg atcttctaca acctgatcaa 2401 accgtacctg atcccgcaga tgatgtacaa actgccgaac actatctcct ccgaaacttt 2461 cctgaaataa gggcccgacg tctgcaggca tgctaaggag gttataaaaa atggatatta 2521 atactgaaac tgagatcaag caaaagcatt cactaacccc ctttcctgtt ttcctaatca 2581 gcccggcatt tcgcgggcga tattttcaca gctatttcag gagttcagcc atgaacgctt 2641 attacattca ggatcgtctt gaggctcaga gctgggcgcg tcactaccag cagctcgccc 2701 gtgaagagaa agaggcagaa ctggcagacg acatggaaaa aggcctgccc cagcacctgt 2761 ttgaatcgct atgcatcgat catttgcaac gccacggggc cagcaaaaaa tccattaccc 2821 gtgcgtttga tgacgatgtt gagtttcagg agcgcatggc agaacacatc cggtacatgg 2881 ttgaaaccat tgctcaccac caggttgata ttgattcaga ggtataaaac gaatgagtac 2941 tgcactcgca acgctggctg ggaagctggc tgaacgtgtc ggcatggatt ctgtcgaccc 3001 acaggaactg atcaccactc ttcgccagac ggcatttaaa ggtgatgcca gcgatgcgca 3061 gttcatcgca ttactgatcg ttgccaacca gtacggcctt aatccgtgga cgaaagaaat 3121 ttacgccttt cctgataagc agaatggcat cgttccggtg gtgggcgttg atggctggtc 3181 ccgcatcatc aatgaaaacc agcagtttga tggcatggac tttgagcagg acaatgaatc 3241 ctgtacatgc cggatttacc gcaaggaccg taatcatccg atctgcgtta ccgaatggat 3301 ggatgaatgc cgccgcgaac cattcaaaac tcgcgaaggc agagaaatca cggggccgtg 3361 gcagtcgcat cccaaacgga tgttacgtca taaagccatg attcagtgtg cccgtctggc 3421 cttcggattt gctggtatct atgacaagga tgaagccgag cgcattgtcg aaaatactgc 3481 atacactgca gaacgtcagc cggaacgcga catcactccg gttaacgatg aaaccatgca 3541 ggagattaac actctgctga tcgccctgga taaaacatgg gatgacgact tattgccgct 3601 ctgttcccag atatttcgcc gcgacattcg tgcatcgtca gaactgacac aggccgaagc 3661 agtaaaagct cttggattcc tgaaacagaa agccgcagag cagaaggtgg cagcatgaca 3721 ccggacatta tcctgcagcg taccgggatc gatgtgagag ctgtcgaaca gggggatgat 3781 gcgtggcaca aattacggct cggcgtcatc accgcttcag aagttcacaa cgtgatagca 3841 aaaccccgct ccggaaagaa gtggcctgac atgaaaatgt cctacttcca caccctgctt 3901 gctgaggttt gcaccggtgt ggctccggaa gttaacgcta aagcactggc ctggggaaaa 3961 cagtacgaga acgacgccag aaccctgttt gaattcactt ccggcgtgaa tgttactgaa 4021 tccccgatca tctatcgcga cgaaagtatg cgtaccgcct gctctcccga tggtttatgc 4081 agtgacggca acggccttga actgaaatgc ccgtttacct cccgggattt catgaagttc 4141 cggctcggtg gtttcgaggc cataaagtca gcttacatgg cccaggtgca gtacagcatg 4201 tgggtgacgc gaaaaaatgc ctggtacttt gccaactatg acccgcgtat gaagcgtgaa 4261 ggcctgcatt atgtcgtgat tgagcgggat gaaaagtaca tggcgagttt tgacgagatc 4321 gtgccggagt tcatcgaaaa aatggacgag gcactggctg aaattggttt tgtatttggg 4381 gagcaatggc gatgacgcat cctcacgata atatccgggt aggcgcaatc actttcgtct 4441 actccgttac aaagcgaggc tgggtatttc ccggcctttc tgttatccga aatccactga 4501 aagcacagcg gctggctgag gagataaata ataaacgagg ggctgtatgc acaaagcatc 4561 ttctgttgag ttaagaacga gtatcgagat ggcacatagc cttgctcaaa ttggaatcag 4621 gtttgtgcca ataccagtag aaacagacga agaatgcatg caagcttggc tgttttggcg 4681 gatgagagaa gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa 4741 aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag 4801 aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact 4861 gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 4921 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt 4981 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa 5041 attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt 5101 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 5161 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 5221 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 5281 agatgctgaa gatcagttgg gtgcacctgc cattcatccg cttattatca cttattcagg 5341 cgtagcacca ggcgtttaag ggcaccaata actgccttaa aaaaattacg ccccgccctg 5401 ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca 5461 gacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata 5521 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa 5581 actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt 5641 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa 5701 ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg 5761 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc 5821 catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata 5881 aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt 5941 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca 6001 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc 6061 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg 6121 aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg 6181 cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac 6241 aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttactga tttagtgtat 6301 gatggtgttt ttgaggtgct ccagtggctt ctgtttctat cagctgtccc tcctgttcag 6361 ctactgacgg ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg 6421 tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc atgtggcagg 6481 agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat acaggatata ttccgcttcc 6541 tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa 6601 cggggcggag atttcctgga agatgccagg aagatactta acagggaagt gagagggccg 6661 cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct 6721 caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg 6781 gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat 6841 ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag 6901 ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat 6961 cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat 7021 tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag 7081 ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag 7141 aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg 7201 cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc 7261 agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta 7321 attctcatgt ttgacagctt atcatcg //