LOCUS pNEB205a 2710 bp DNA circular 18-OCT-2007 DEFINITION Cloning vector pNEB205a, complete sequence. ACCESSION VERSION KEYWORDS . SOURCE Cloning vector pNEB205a ORGANISM Cloning vector pNEB205a other sequences; artificial sequences; vectors. REFERENCE 1 (bases 1 to 2710) AUTHORS Bitinaite,J., Rubino,M., Varma,K.H., Schildkraut,I., Vaisvila,R. and Vaiskunaite,R. TITLE USER friendly DNA engineering and cloning method by uracil excision JOURNAL Nucleic Acids Res 35 (6), 1992-2002 (2007) PUBMED 17341463 REFERENCE 2 (bases 1 to 2710) AUTHORS Bitinaite,J. TITLE Direct Submission JOURNAL Submitted (18-OCT-2007) Research Department, New England Biolabs, 240 County Road, Ipswich, MA 01938, USA COMMENT The plasmid is supplied in a linearized form 2706 bp in length (with bp 431..437 excised from the circular form), including two flanking, noncomplementary 8-base 3' overhangs at the intended cloning site. This form consists of bases (438..2710,1..430) where 430..437 and 442..449 are single stranded. FEATURES Location/Qualifiers source 1..2710 /organism="Cloning vector pNEB205a" /mol_type="other DNA" gene complement(146..493) /gene="lacZalpha" CDS complement(146..493) /gene="lacZalpha" /codon_start=1 /product="beta-galactosidase alpha fragment" /translation="MTMITPSFLQGLNAEETSRLSLSLIKARLSSNSLAVVLQRRDWE NPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHR IWCTLSTICSDAA" misc_feature 396..476 /gene="lacZalpha" /note="multiple cloning site (EcoRI-HindIII); top strand nt 438-449 and bottom strand nt 430-441 deleted by XbaI + N.BbvCIB digestion" -10_signal complement(538..543) /note="Plac promoter (drives lacZalpha; counter-clockwise); TATGTT" -35_signal complement(562..567) /note="Plac promoter (drives lacZalpha; counter-clockwise); TTTACA" protein_bind complement(587..599) /note="CAP protein binding site" /bound_moiety="catabolite activator protein (CAP)" rep_origin complement(891..1479) /note="pMB1 origin of replication (counter-clockwise) (RNAII -35 to RNA/DNA switch point)" misc_RNA complement(891..1443) /note="RNAII transcript (complementary strand)" -35_signal 1297..1302 /note="RNAI promoter (clockwise); TTGAAG" -10_signal 1319..1324 /note="RNAI promoter (clockwise); GCTACA" misc_RNA 1333..1440 /note="RNAI transcript" -10_signal complement(1453..1458) /note="RNAII promoter (counter-clockwise); CGTAAT" -35_signal complement(1474..1479) /note="RNAII promoter (counter-clockwise); TTGAGA" gene complement(1650..2510) /gene="bla" CDS complement(1650..2510) /gene="bla" /note="ampR (confers resistance to ampicillin)" /codon_start=1 /product="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" sig_peptide complement(2442..2510) /gene="bla" /note="required for secretion to the periplasm; cleaved off to form the mature beta-lactamase protein." -10_signal complement(2554..2559) /note="bla promoter (counter-clockwise); GAGACA; transcript start 2545 (complementary strand)" -35_signal complement(2575..2580) /note="bla promoter (counter-clockwise); TTCAAA" BASE COUNT 676 a 678 c 689 g 667 t ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcagg cgcgccttaa 421 ttaagctgag ggaaagtcta gatgtctcct cagcgtttaa accctgcagg aagcttggcg 481 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 541 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 601 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 661 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 721 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 781 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 841 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 901 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 961 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 1021 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 1081 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1141 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1201 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1261 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1321 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1381 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1441 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1501 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1561 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1621 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1681 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1741 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1801 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1861 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1921 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1981 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 2041 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 2101 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2161 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2221 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2281 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2341 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2401 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2461 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2521 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2581 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2641 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 2701 ccctttcgtc //