LOCUS pNBio 6895 bp ds-DNA circular 12-JUN-2008 DEFINITION Mammalian expression vector pNBio, complete sequence. ACCESSION DQ520290 VERSION DQ520290.1 GI:104744457 KEYWORDS . SOURCE Mammalian expression vector pNBio ORGANISM Mammalian expression vector pNBio other sequences; artificial sequences; vectors. REFERENCE 1 (bases 1 to 6895) AUTHORS Kulman,J.D., Satake,M. and Harris,J.E. TITLE A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells JOURNAL Protein Expr. Purif. 52 (2), 320-328 (2007) PUBMED 17084093 REFERENCE 2 (bases 1 to 6895) AUTHORS Kulman,J.D., Satake,M. and Harris,J.E. TITLE Direct Submission JOURNAL Submitted (28-APR-2006) Biochemistry, University of Washington, N.E. Pacific St., Seattle, WA 98195, USA COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers source join(1..651,1239..2332,3501..5562) /organism="Mammalian expression vector pNBio" /mol_type="other DNA" /db_xref="taxon:386594" /label=source:Mammalian expression vector pNBio /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink source 5898..6722 /organism="Mammalian expression vector pNBioSec" /mol_type="other DNA" /db_xref="taxon:478810" /label=source:Mammalian expression vector pNBioSec /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink misc_feature 5563..5793 /label=SV40 enhancer /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 promoter 7..439 /note="tetracycline-responsive promoter" /label=tetracycline-responsive promoter /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 protein_bind 15..33 /note="tetO repeat" /bound_moiety="TetR" /label=tetO repeat /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink protein_bind 57..75 /note="tetO repeat" /bound_moiety="TetR" /label=tetO repeat(1) /ApEinfo_label=tetO repeat /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink protein_bind 99..117 /note="tetO repeat" /bound_moiety="TetR" /label=tetO repeat(2) /ApEinfo_label=tetO repeat /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink protein_bind 141..159 /note="tetO repeat" /bound_moiety="TetR" /label=tetO repeat(3) /ApEinfo_label=tetO repeat /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink protein_bind 183..201 /note="tetO repeat" /bound_moiety="TetR" /label=tetO repeat(4) /ApEinfo_label=tetO repeat /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink protein_bind 225..243 /note="tetO repeat" /bound_moiety="TetR" /label=tetO repeat(5) /ApEinfo_label=tetO repeat /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink protein_bind 267..285 /note="tetO repeat" /bound_moiety="TetR" /label=tetO repeat(6) /ApEinfo_label=tetO repeat /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink promoter 320..439 /note="minimal CMV promoter" /label=minimal CMV promoter /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 TATA_signal 342..349 /label=TATA_signal /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink RBS 477..485 /note="Kozak site" /label=Kozak site /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink misc_feature 492..530 /note="biotin acceptor peptide (BioTag)-encoding" /label=biotin acceptor peptide (BioTag)-encoding /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 532..551 /note="multiple cloning site; MCS" /label=multiple cloning site; MCS /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 645..651 /note="encephalomyocarditis virus internal ribosome entry site; IRES" /label=encephalomyocarditis virus internal ribosome entry /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff gene 1270..2283 /gene="birA" /label=birA /ApEinfo_fwdcolor=#00ff6e /ApEinfo_revcolor=#00ff6e RBS 1270..1278 /gene="birA" /note="Kozak site" /label=birA(1) /ApEinfo_label=birA /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink misc_feature 1282..1305 /gene="birA" /note="FLAG epitope tag-encoding" /label=birA(2) /ApEinfo_label=birA /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 1318..2283 /gene="birA" /note="Escherichia coli BirA (biotin ligase)" /label=birA(3) /ApEinfo_label=birA /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff rep_origin 3702..4345 /note="Col E1 origin of replication" /label=Col E1 origin of replication /ApEinfo_fwdcolor=#00c0cb /ApEinfo_revcolor=#00c0cb gene complement(4493..5353) /gene="Ampr" /label=Ampr /ApEinfo_fwdcolor=#00ff6e /ApEinfo_revcolor=#00ff6e CDS complement(4493..5353) /gene="Ampr" /note="ampicillin resistance" /codon_start=1 /product="beta-lactamase" /protein_id="ABF74575.1" /db_xref="GI:104744459" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" /label=beta-lactamase /ApEinfo_fwdcolor=#00ff00 /ApEinfo_revcolor=#00ff00 promoter 5898..5984 /label=promoter /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 misc_feature 6723..6895 /label=inverted SV40 late poly(A) signal /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan misc_feature 652..1238 /note="encephalomyocarditis virus internal ribosome entry site; IRES" /label=EMCV-R IRES /ApEinfo_fwdcolor=#00fff8 /ApEinfo_revcolor=#00fff8 misc_feature 5844..5897 /label=SV40 Promoter /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 polyA_signal 2333..3500 /note="beta-globin poly-A signal" /label=beta-globin poly-A signal /ApEinfo_fwdcolor=#ff00cb /ApEinfo_revcolor=#ff00cb misc_feature 5794..5843 /label=SV40 Promoter(1) /ApEinfo_label=SV40 Promoter /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 misc_feature 5794..5843 /label=SV40 transcription start sites region /ApEinfo_fwdcolor=#ffb1be /ApEinfo_revcolor=#ffb1be misc_feature 5794..5794 /label=SV40 transcription start (+1) /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 5832..5832 /label=SV40 transcription start (+1)(1) /ApEinfo_label=SV40 transcription start (+1) /ApEinfo_fwdcolor=#fe00ff /ApEinfo_revcolor=#fe00ff CDS 1279..2283 /gene="birA" /note="holocarboxylase synthetase" /codon_start=1 /product="biotin ligase" /protein_id="ABF74574.1" /db_xref="GI:104744458" /translation="MDYKDDDDKSPRSMKDNTVPLKLIALLANGEFHSGEQLGETLGM SRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDS TNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAI GLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGA GINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLS RWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK " /label=biotin ligase /ApEinfo_fwdcolor=#00ff00 /ApEinfo_revcolor=#00ff00 misc_feature 5838..5838 /label=SV40 transcription start (+1)(2) /ApEinfo_label=SV40 transcription start (+1) /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 5843..5843 /label=SV40 transcription start (+1)(3) /ApEinfo_label=SV40 transcription start (+1) /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff CDS 5985..6584 /codon_start=1 /transl_table=11 /product="puromycin N-acetyltransferase" /protein_id="ABW71928.1" /db_xref="GI:158534339" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSR LAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" /label=puromycin N-acetyltransferase /ApEinfo_fwdcolor=#00ff00 /ApEinfo_revcolor=#00ff00 misc_feature 371..371 /label=putative CMV transcription start site (+1) /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 3141..3141 /label=poly(A) tail appended after this base /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan ORIGIN 1 ctcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc 61 tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa 121 gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc 181 actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag 241 agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag 301 ctcggtaccc gggtcgaggt aggcgtgtac ggtgggaggc ctatataagc agagctcgtt 361 tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac 421 accgggaccg atccagcctc cgcggccccg aattcgagct cggtacccgg ggatctgccg 481 ccaccatggg cctgaacgac atcttcgaag ctcagaaaat cgaatggcac gggatccgat 541 atcgcggccg cttgaacgac atttttgaag cgcaaaaaat tgaatggcat taaggccgcg 601 ctagcctcga gaattcacgc gtcgagcatg catctagggc ggccaattcc gcccctctcc 661 ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt 721 ctatatgtga ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg 781 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg 841 tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc 901 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca 961 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag 1021 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa 1081 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt 1141 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 1201 tttcctttga aaaacacgat gataagcttg ccacaaccca aacttaagct tggtaccgag 1261 ctcggatctg ccgccaccat ggattacaag gatgacgacg ataagagccc gagatccatg 1321 aaggataaca ccgtgccact gaaattgatt gccctgttag cgaacggtga atttcactct 1381 ggcgagcagt tgggtgaaac gctgggaatg agccgggcgg ctattaataa acacattcag 1441 acactgcgtg actggggcgt tgatgtcttt accgttccgg gtaaaggata cagcctgcct 1501 gagcctatcc agttacttaa tgctaaacag atattgggtc agctggatgg cggtagtgta 1561 gccgtgctgc cagtgattga ctccacgaat cagtaccttc ttgatcgtat cggagagctt 1621 aaatcgggcg atgcttgcat tgcagaatac cagcaggctg gccgtggtcg ccggggtcgg 1681 aaatggtttt cgccttttgg cgcaaactta tatttgtcga tgttctggcg tctggaacaa 1741 ggcccggcgg cggcgattgg tttaagtctg gttatcggta tcgtgatggc ggaagtatta 1801 cgcaagctgg gtgcagataa agttcgtgtt aaatggccta atgacctcta tctgcaggat 1861 cgcaagctgg caggcattct ggtggagctg actggcaaaa ctggcgatgc ggcgcaaata 1921 gtcattggag ccgggatcaa catggcaatg cgccgtgttg aagagagtgt cgttaatcag 1981 gggtggatca cgctgcagga agcggggatc aatctcgatc gtaatacgtt ggcggccatg 2041 ctaatacgtg aattacgtgc tgcgttggaa ctcttcgaac aagaaggatt ggcaccttat 2101 ctgtcgcgct gggaaaagct ggataatttt attaatcgcc cagtgaaact tatcattggt 2161 gataaagaaa tatttggcat ttcacgcgga atagacaaac agggggcttt attacttgag 2221 caggatggaa taataaaacc ctggatgggc ggtgaaatat ccctgcgtag tgcagaaaaa 2281 taagaattca gatctctgca gctcgagtct agagggcccg tttatctcta gagctgagaa 2341 cttcagggtg agtttgggga cccttgattg ttctttcttt ttcgctattg taaaattcat 2401 gttatatgga gggggcaaag ttttcagggt gttgtttaga atgggaagat gtcccttgta 2461 tcaccatgga ccctcatgat aattttgttt ctttcacttt ctactctgtt gacaaccatt 2521 gtctcctctt attttctttt cattttctgt aacttttttc gttaaacttt agcttgcatt 2581 tgtaacgaat ttttaaattc actttcgttt atttgtcaga ttgtaagtac tttctctaat 2641 cacttttttt tcaaggcaat cagggtaatt atattgtact tcagcacagt tttagagaac 2701 aattgttata attaaatgat aaggtagaat atttctgcat ataaattctg gctggcgtgg 2761 aaatattctt attggtagaa acaactacat cctggtaatc atcctgcctt tctctttatg 2821 gttacaatga tatacactgt ttgagatgag gataaaatac tctgagtcca aaccgggccc 2881 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt 2941 tgttgtgctg tctcatcatt ttggcaaaga attcactcct caggtgcagg ctgcctatca 3001 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc 3061 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctgggtaata 3121 aaggaaattt attttcattg caatagtgtg tgggaatttt ttgtgtctct cactcggaag 3181 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc 3241 aacatatgcc atatgctggc tgccatgaac aaaggtggct ataaagaggt catcagtata 3301 tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga 3361 ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac 3421 atgttttact agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc 3481 ttctcttatg aactcgactg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 3541 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 3601 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 3661 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 3721 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3781 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 3841 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 3901 tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt 3961 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 4021 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 4081 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 4141 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 4201 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 4261 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 4321 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 4381 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 4441 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 4501 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 4561 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 4621 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 4681 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 4741 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 4801 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 4861 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 4921 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 4981 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 5041 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 5101 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 5161 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 5221 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 5281 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 5341 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 5401 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 5461 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct 5521 ataaaaatag gcgtatcacg aggccctttc gtcttcactc gagtgtgtca gttagggtgt 5581 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 5641 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 5701 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 5761 cccagttccg cccattctcc gccccatggc tgaCTAATTT TTTTTATTTA TGCAGAGGCC 5821 GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA 5881 GGCTTTTGCA AAAAGCTtgc atgcctgcag gtcggccgcc acgaccggtg ccgccaccat 5941 cccctgaccc acgcccctga cccctcacaa ggagacgacc ttccatgacc gagtacaagc 6001 ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg 6061 cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg 6121 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg 6181 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg 6241 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc 6301 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg 6361 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc 6421 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc 6481 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg 6541 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc 6601 cgcagcgccc gaccgaaagg agcgcacgac cccatggctc cgaccgaagc cgacccgggc 6661 ggccccgccg accccgcacc cgcccccgag gcccaccgac tctagaggat cataatcagc 6721 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac 6781 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt 6841 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgc //