LOCUS pNBioSec 7032 bp ds-DNA circular 12-JUN-2008 DEFINITION Mammalian expression vector pNBioSec, complete sequence. ACCESSION EU082004 VERSION EU082004.1 GI:158534336 KEYWORDS . SOURCE Mammalian expression vector pNBioSec ORGANISM Mammalian expression vector pNBioSec other sequences; artificial sequences; vectors. REFERENCE 1 (bases 1 to 7032) AUTHORS Mize,G.J., Harris,J.E., Takayama,T.K. and Kulman,J.D. TITLE Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells JOURNAL Protein Expr. Purif. 57 (2), 280-289 (2008) PUBMED 18042400 REFERENCE 2 (bases 1 to 7032) AUTHORS Mize,G.J., Harris,J.E., Takayama,T.K. and Kulman,J.D. TITLE Direct Submission JOURNAL Submitted (04-AUG-2007) Department of Biochemistry, University of Washington, N.E. Pacific Street, Seattle, WA 98195, USA COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers source join(1..728,1316..2469,3638..5699,6035..6859) /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 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 misc_feature 5931..5980 /label=SV40 Promoter /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 misc_feature 5931..5980 /label=SV40 transcription start sites region /ApEinfo_fwdcolor=#ffb1be /ApEinfo_revcolor=#ffb1be misc_feature 5975..5975 /label=SV40 transcription start (+1) /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff protein_bind 141..159 /note="tetO repeat" /bound_moiety="TetR" /label=tetO repeat(3) /ApEinfo_label=tetO repeat /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink misc_feature 5981..6034 /label=SV40 Promoter(1) /ApEinfo_label=SV40 Promoter /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 misc_feature 5931..5931 /label=SV40 transcription start (+1)(1) /ApEinfo_label=SV40 transcription start (+1) /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 5969..5969 /label=SV40 transcription start (+1)(2) /ApEinfo_label=SV40 transcription start (+1) /ApEinfo_fwdcolor=#fe00ff /ApEinfo_revcolor=#fe00ff 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 570..608 /note="biotin acceptor peptide (BioTag)-encoding" /label=biotin acceptor peptide (BioTag)-encoding /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 609..628 /note="multiple cloning site; MCS" /label=multiple cloning site; MCS /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff RBS 1326..1334 /note="Kozak site" /label=Kozak site(1) /ApEinfo_label=Kozak site /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink CDS 1335..2420 /note="holocarboxylase synthetase; secreted variant of Escherichia coli biotin ligase with an amino-terminal extension consisting of the bovine prolactin signal peptide and a Flag epitope tag" /codon_start=1 /transl_table=11 /product="BirA" /protein_id="ABW71926.1" /db_xref="GI:158534337" /translation="MDSKGSSQKGSRLLLLLVVSNLLLCQGVVSDYKDDDDKLKMKDN TVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPE PIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRG RKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLY LQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRN TLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDK QGALLLEQDGIIKPWMGGEISLRSAEK" /label=BirA /ApEinfo_fwdcolor=#00ff00 /ApEinfo_revcolor=#00ff00 sig_peptide 1335..1424 /note="bovine prolactin signal peptide" /label=bovine prolactin signal peptide /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink misc_feature 1425..1448 /note="Region: Flag epitope tag" /label=Region: Flag epitope tag /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 1452..2417 /note="native coding sequence of E. coli BirA; Region: biotin ligase" /label=native coding sequence of E. coli BirA; Region: bi /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff rep_origin 3839..4482 /note="Col E1 origin of replication" /label=Col E1 origin of replication /ApEinfo_fwdcolor=#00c0cb /ApEinfo_revcolor=#00c0cb CDS complement(4630..5490) /codon_start=1 /transl_table=11 /product="beta-lactamase" /protein_id="ABW71927.1" /db_xref="GI:158534338" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" /label=beta-lactamase /ApEinfo_fwdcolor=#00ff00 /ApEinfo_revcolor=#00ff00 promoter 6035..6121 /label=promoter /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 CDS 6122..6721 /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 polyA_signal 2470..3637 /note="beta-globin poly-A signal" /label=beta-globin poly-A signal /ApEinfo_fwdcolor=#ff00cb /ApEinfo_revcolor=#ff00cb misc_feature 6860..7032 /label=inverted SV40 late poly(A) signal /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan misc_feature 371..371 /label=putative CMV transcription start site (+1) /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 729..1315 /note="encephalomyocarditis virus internal ribosome entry site; IRES" /label=EMCV-R IRES /ApEinfo_fwdcolor=#00fff8 /ApEinfo_revcolor=#00fff8 misc_feature 5700..5930 /label=SV40 enhancer /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 misc_feature 5980..5980 /label=SV40 transcription start (+1)(3) /ApEinfo_label=SV40 transcription start (+1) /ApEinfo_fwdcolor=#ff00ff /ApEinfo_revcolor=#ff00ff misc_feature 3278..3278 /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 ccaccatgcc gtcttctgtc tcgtggggca tcctcctgct ggcaggcctg tgctgcctgg 541 tccctgtctc cctggctgag cgtggatctt tgaacgacat ttttgaagcg caaaaaattg 601 aatggcatgg atccgatatc gcggccgctt gaacgacatt tttgaagcgc aaaaaattga 661 atggcattaa ggccgcgcta gcctcgagaa ttcacgcgtc gagcatgcat ctagggcggc 721 caattccgcc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa 781 ggccggtgtg cgtttgtcta tatgtgattt tccaccatat tgccgtcttt tggcaatgtg 841 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc 901 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct 961 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac 1021 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc 1081 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta 1141 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg 1201 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg 1261 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aagcttgcca caacccaaac 1321 ttaaggccgc caccatggac agcaaaggtt cgtcgcagaa agggtcccgc ctgctcctgc 1381 tgctggtggt gtcaaatcta ctcttgtgcc agggtgtggt ctccgattat aaggatgatg 1441 atgataagct taagatgaag gataacaccg tgccactgaa attgattgcc ctgttagcga 1501 acggtgaatt tcactctggc gagcagttgg gtgaaacgct gggaatgagc cgggcggcta 1561 ttaataaaca cattcagaca ctgcgtgact ggggcgttga tgtctttacc gttccgggta 1621 aaggatacag cctgcctgag cctatccagt tacttaatgc taaacagata ttgggtcagc 1681 tggatggcgg tagtgtagcc gtgctgccag tgattgactc cacgaatcag taccttcttg 1741 atcgtatcgg agagcttaaa tcgggcgatg cttgcattgc agaataccag caggctggcc 1801 gtggtcgccg gggtcggaaa tggttttcgc cttttggcgc aaacttatat ttgtcgatgt 1861 tctggcgtct ggaacaaggc ccggcggcgg cgattggttt aagtctggtt atcggtatcg 1921 tgatggcgga agtattacgc aagctgggtg cagataaagt tcgtgttaaa tggcctaatg 1981 acctctatct gcaggatcgc aagctggcag gcattctggt ggagctgact ggcaaaactg 2041 gcgatgcggc gcaaatagtc attggagccg ggatcaacat ggcaatgcgc cgtgttgaag 2101 agagtgtcgt taatcagggg tggatcacgc tgcaggaagc ggggatcaat ctcgatcgta 2161 atacgttggc ggccatgcta atacgtgaat tacgtgctgc gttggaactc ttcgaacaag 2221 aaggattggc accttatctg tcgcgctggg aaaagctgga taattttatt aatcgcccag 2281 tgaaacttat cattggtgat aaagaaatat ttggcatttc acgcggaata gacaaacagg 2341 gggctttatt acttgagcag gatggaataa taaaaccctg gatgggcggt gaaatatccc 2401 tgcgtagtgc agaaaaataa gaattcagat ctctgcagct cgagtctaga gggcccgttt 2461 atctctagag ctgagaactt cagggtgagt ttggggaccc ttgattgttc tttctttttc 2521 gctattgtaa aattcatgtt atatggaggg ggcaaagttt tcagggtgtt gtttagaatg 2581 ggaagatgtc ccttgtatca ccatggaccc tcatgataat tttgtttctt tcactttcta 2641 ctctgttgac aaccattgtc tcctcttatt ttcttttcat tttctgtaac ttttttcgtt 2701 aaactttagc ttgcatttgt aacgaatttt taaattcact ttcgtttatt tgtcagattg 2761 taagtacttt ctctaatcac ttttttttca aggcaatcag ggtaattata ttgtacttca 2821 gcacagtttt agagaacaat tgttataatt aaatgataag gtagaatatt tctgcatata 2881 aattctggct ggcgtggaaa tattcttatt ggtagaaaca actacatcct ggtaatcatc 2941 ctgcctttct ctttatggtt acaatgatat acactgtttg agatgaggat aaaatactct 3001 gagtccaaac cgggcccctc tgctaaccat gttcatgcct tcttcttttt cctacagctc 3061 ctgggcaacg tgctggttgt tgtgctgtct catcattttg gcaaagaatt cactcctcag 3121 gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac 3181 cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat 3241 ctgacttctg ggtaataaag gaaatttatt ttcattgcaa tagtgtgtgg gaattttttg 3301 tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat 3361 ttggtttaga gtttggcaac atatgccata tgctggctgc catgaacaaa ggtggctata 3421 aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc 3481 cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc 3541 cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc 3601 agtcatagct gtccctcttc tcttatgaac tcgactgcat taatgaatcg gccaacgcgc 3661 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 3721 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 3781 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 3841 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 3901 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 3961 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 4021 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 4081 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 4141 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 4201 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 4261 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 4321 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 4381 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 4441 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 4501 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 4561 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 4621 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 4681 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 4741 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 4801 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 4861 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 4921 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 4981 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 5041 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 5101 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 5161 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 5221 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 5281 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 5341 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 5401 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 5461 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 5521 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 5581 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 5641 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcactcgag 5701 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 5761 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 5821 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 5881 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga CTAATTTTTT 5941 TTATTTATGC AGAGGCCGAG GCCGCCTCGG CCTCTGAGCT ATTCCAGAAG TAGTGAGGAG 6001 GCTTTTTTGG AGGCCTAGGC TTTTGCAAAA AGCTtgcatg cctgcaggtc ggccgccacg 6061 accggtgccg ccaccatccc ctgacccacg cccctgaccc ctcacaagga gacgaccttc 6121 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt 6181 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga 6241 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga 6301 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga 6361 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg 6421 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga 6481 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg 6541 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct 6601 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc 6661 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg 6721 acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atggctccga 6781 ccgaagccga cccgggcggc cccgccgacc ccgcacccgc ccccgaggcc caccgactct 6841 agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 6901 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 6961 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 7021 ttttttcact gc //