LOCUS Exported 8599 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION enhance solubility and correct disulfide bond-formation of fused protein expressed in E.coli. ACCESSION . VERSION . KEYWORDS pDEST-hPDI SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8599) AUTHORS Song JA, Koo BK, Chong SH, Kwak J, Ryu HB, Nguyen MT, Vu TT, Jeong B, Kim SW, Choe H TITLE Expression and purification of biologically active human FGF2 containing the b'a' domains of human PDI in Escherichia coli. JOURNAL Appl Biochem Biotechnol. 2013 May;170(1):67-80. doi: 10.1007/s12010-013-0140-3. Epub 2013 Mar 8. PUBMED 23471584 REFERENCE 2 (bases 1 to 8599) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8599 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 326..631 /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 510..529 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(672..796) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 810..827 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(880..898) /label=T7 Term /note="T7 terminator, reverse primer" terminator 894..941 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 978..1433 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1065..1084) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1275..1296 /label=F1ori-F /note="F1 origin, forward primer" promoter 1460..1564 /gene="bla" /label=AmpR promoter CDS 1565..2425 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1783..1802) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2596..3184 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3085..3104 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3338..3355 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3370..3512 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3438..3457) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3598..3620 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3614..3805) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4614..5696) /codon_start=1 /gene="lacI" /product="lac repressor" /label=lacI /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 5658..5677 /label=LacI-R /note="LacI, reverse primer" promoter complement(5697..5774) /gene="lacI" /label=lacI promoter primer_bind 5980..5999 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 6089..6108 /label=T7 /note="T7 promoter, forward primer" promoter 6089..6107 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 6108..6132 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." RBS 6147..6169 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 6182..6205 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" protein_bind 7691..7815 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 7840..7870 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 7924..8583 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(7990..8009) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" ORIGIN 1 tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat 61 atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc agcgtattac 121 agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc 181 ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa 241 gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt 301 gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag 361 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat 421 ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc 481 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc 541 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa 601 cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc 661 tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt 721 tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt 781 tcttgtacaa agtggtgatg tacctcgagc accaccacca ccaccactga gatccggctg 841 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat 901 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat 961 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 1021 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 1081 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 1141 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 1201 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 1261 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 1321 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 1381 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcaggtgg 1441 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 1501 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 1561 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 1621 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 1681 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 1741 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 1801 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 1861 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 1921 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 1981 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 2041 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 2101 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 2161 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 2221 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 2281 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 2341 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 2401 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 2461 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 2521 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 2581 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 2641 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 2701 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 2761 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 2821 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 2881 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 2941 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 3001 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 3061 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 3121 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 3181 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 3241 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 3301 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 3361 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 3421 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc 3481 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg 3541 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 3601 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt 3661 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca 3721 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa 3781 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg 3841 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac 3901 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc 3961 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac 4021 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact 4081 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg 4141 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct 4201 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca 4261 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg 4321 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg 4381 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg 4441 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga 4501 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg 4561 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc 4621 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 4681 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc 4741 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg 4801 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag 4861 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac 4921 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg 4981 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag 5041 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca 5101 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg 5161 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata 5221 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag 5281 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg 5341 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct 5401 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca 5461 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt 5521 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct 5581 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg 5641 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc 5701 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc 5761 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca 5821 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga 5881 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc 5941 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc 6001 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat 6061 cgagatccag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg 6121 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatgaa 6181 acaccatcac catcaccatc accatgagct cgacgcaccg gaagaagagg atcatgtcct 6241 ggttctgcgc aaaagcaact tcgcggaagc actggccgca cataaatatc tgctggtgga 6301 attttatgct ccttggtgcg gtcattgcaa agccctggcc ccggagtacg ccaaagccgc 6361 aggtaaactg aaagctgaag gtagcgaaat ccgcctggca aaggttgatg ctacggaaga 6421 gagtgacctg gcgcagcagt atggtgtccg cggctatcct acaattaaat tcttccgtaa 6481 cggtgatacc gcatctccaa aagaatatac cgctggtcgc gaggcggacg atattgttaa 6541 ctggctgaag aaacgcactg gccctgccgc aaccaccctg cctgatggcg ctgctgccga 6601 aagcctggtc gaaagtagcg aagttgccgt cattggtttc tttaaggatg tagaatctga 6661 cagtgccaaa cagtttctgc aagcggcaga ggctatcgat gacatcccgt tcggcatcac 6721 ctctaacagt gacgtattca gtaaatacca actggataaa gacggcgttg tgctgttcaa 6781 gaaatttgat gaaggtcgca acaactttga gggtgaggtg accaaggaga acctgctgga 6841 ttttattaag cacaaccaac tgccgctggt tattgaattt acagaacaaa cggcgccgaa 6901 aattttcggc ggtgagatta aaacacatat cctgctgttt ctgccgaaga gcgtttctga 6961 ttacgatggt aaactgagta attttaaaac cgccgcagaa tctttcaaag gtaagattct 7021 gttcattttc attgatagcg accacacgga caatcagcgt atcctggagt tctttggtct 7081 gaagaaagag gaatgcccgg ctgtgcgtct gattacgctg gaagaggaaa tgacaaagta 7141 caagccggag agcgaggaac tgactgcaga acgtatcacc gaattttgtc atcgtttcct 7201 ggaggggaag attaagccgc atctgatgag ccaggaactg ccggaggatt gggacaaaca 7261 gccagtgaaa gttctggtgg ggaagaattt tgaagatgtg gccttcgatg agaagaagaa 7321 tgtgtttgtg gagttctacg ccccgtggtg tgggcactgt aaacagctgg cgccgatctg 7381 ggacaaactg ggcgaaacgt ataaagatca cgaaaatatt gtgatcgcga aaatggattc 7441 taccgcgaat gaagtagaag cggtaaaagt acactctttt ccgacgctga aattctttcc 7501 agcgagcgcg gatcgtactg tcattgatta taatggcgaa cgtactctgg acggctttaa 7561 gaaatttctg gaaagcggcg gccaggatgg cgcgggcgat gatgatgacc tggaagacct 7621 ggaagaagcg gaggaaccag acatggagga ggatgacgac cagaaagcgg tcggtaccgg 7681 atcttacatc acaagtttgt acaaaaaagc tgaacgagaa acgtaaaatg atataaatat 7741 caatatatta aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca 7801 tatccagtca ctatggcggc cgcattaggc accccaggct ttacacttta tgcttccggc 7861 tcgtataatg tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct 7921 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa 7981 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg 8041 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt 8101 attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac 8161 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact 8221 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata 8281 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt 8341 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac 8401 gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa 8461 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc 8521 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg 8581 taaacgcgtg gatccggct //