LOCUS Exported 6508 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION FX cloning E.coli expression vector with araBAD promoter and C-terminal 3C protease cleavage site, GFP and 10x His tags. ACCESSION . VERSION . KEYWORDS pBXC3GH SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6508) AUTHORS Geertsma ER, Dutzler R TITLE A versatile and efficient high-throughput cloning tool for structural biology. JOURNAL Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25. PUBMED 21410291 REFERENCE 2 (bases 1 to 6508) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6508 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..285 /gene="araBAD" /label=araBAD promoter /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" primer_bind 208..227 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" CDS complement(370..675) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(472..491) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(1017..1676) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 1591..1610 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(1730..1760) /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind complement(1785..1909) /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS complement(1928..1948) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1952..1972) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1999..2022 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 2029..2739 /codon_start=1 /product="GFP variant that folds robustly even when fused to poorly folded proteins (Pedelacq et al., 2006)" /label=superfolder GFP /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITHGMDELYK" primer_bind complement(2056..2084) /label=GFP-R /note="GFP, reverse primer. Does NOT anneal to EGFP" primer_bind 2679..2699 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" CDS 2746..2775 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 2791..2820 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2836..2853 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2909..2926) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 3079..3165 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 3257..3284 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" promoter 3303..3394 /gene="bla" /label=AmpR promoter CDS 3395..4255 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(3613..3632) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4426..5014 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4915..4934 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5168..5185 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5200..5340 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5266..5285) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5426..5448 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5604..6482) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" ORIGIN 1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggcact 61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc 301 taacaggagg aattaaccat gagttgaaga gcgacctgca gactggctgt gtataaggga 361 gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg 421 gtggctgaga tcagccactt cttccccgat aacggagacc ggcacactgg ccatatcggt 481 ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt cacgggagac 541 tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc cgggcgtgtc 601 aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta 661 aaccttaaac tgcatttcac cagcccctgt tctcgtcagc aaaagagccg ttcatttcaa 721 taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt tcggcacgca 781 gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg acatcatata 841 tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct gcttcatagc 901 atacctcttt ttgacatact tcgggtatac atatcagtat atattcttat accgcaaaaa 961 tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggatcc acgcgtttac 1021 gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg 1081 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct 1141 tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg 1201 tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca 1261 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat 1321 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca 1381 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg 1441 tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata 1501 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc 1561 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct 1621 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta 1681 gcttccttag ctcctgaaaa tctcgacgga tcctaactca aaatccacac attatacgag 1741 ccggaagcat aaagtgtaaa gcctggggtg cctaatgcgg ccgccatagt gactggatat 1801 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga 1861 tatttatatc attttacgtt tctcgttcag cttttttgta caaacttgtg atatcgaatt 1921 cgctagctac ctttctcttc ttttttggat ctacctttct cttctttttt ggggatccac 1981 tagttcagct cttctgcatt agaagttttg tttcaaggtc cacaattcag caaaggagaa 2041 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac 2101 aaattttctg tcagtggaga gggtgaaggt gatgctacat acggaaaact cacccttaaa 2161 tttatttgca ctactggaaa actacctgtt ccttggccaa cacttgtcac tactctgacc 2221 tatggtgttc aatgcttttc ccgttatccg gatcacatga aacggcatga ctttttcaag 2281 agtgccatgc ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggaac 2341 tacaagacgc gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta 2401 aagggtattg attttaaaga agatggaaac attctcggac acaaactaga gtacaactat 2461 aactcacaca atgtatacat cacggcagac aaacaaaaga atggaatcaa agctaacttc 2521 aaaattcgcc acaacattga agatggttcc gttcaactag cagaccatta tcaacaaaat 2581 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc gacacaatct 2641 gccctttcga aagatcccaa cgaaaagcgt gaccacatgg tccttcttga gtttgtaact 2701 gctgctggga ttacacatgg catggatgag ctctacaaaa ctagtcatca tcatcaccat 2761 catcatcacc atcattaagt cgaccatcta gaacaaaaac tcatctcaga agaggatctg 2821 aatagcgccg tcgaccatca tcatcatcat cattgagttt aaacggtctc cagcttggct 2881 gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg 2941 gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc 3001 cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag 3061 tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt 3121 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat 3181 ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc 3241 aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact 3301 cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 3361 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 3421 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 3481 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 3541 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 3601 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact 3661 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 3721 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 3781 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 3841 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 3901 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 3961 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 4021 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 4081 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 4141 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 4201 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 4261 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 4321 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 4381 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 4441 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 4501 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 4561 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 4621 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 4681 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 4741 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 4801 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 4861 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 4921 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 4981 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 5041 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 5101 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 5161 cgagcgcagc gagtcagtga gcgaggaagc ggaagtgccc ctgatgcggt attttctcct 5221 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga 5281 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg 5341 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 5401 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 5461 tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa 5521 tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg 5581 tcaattgtct gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct 5641 tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag 5701 aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg 5761 gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta 5821 atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt 5881 gcgacgctgg cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc 5941 tcgcgtaccc gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc 6001 agtaacaatt gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc 6061 ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg 6121 cgaaagaacc ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg 6181 cgcggacgaa agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag 6241 tgatgaatct ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt 6301 ccctgatttt tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt 6361 cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc 6421 gccaccagat gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc 6481 atacttttca tactcccgcc attcagag //