LOCUS Exported 5498 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION use to create N-terminal PelB and C-terminal Twin-Strep-tagged fusion proteins for periplasmic translocation via PelB signal sequence. ACCESSION . VERSION . KEYWORDS pTDpelB-CTwinStrep SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5498) AUTHORS Dammeyer T, Timmis KN, Tinnefeld P TITLE Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein. JOURNAL Microb Cell Fact. 2013 May 20;12(1):49. PUBMED 23687945 REFERENCE 2 (bases 1 to 5498) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5498 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 5..99 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS 335..1126 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" oriT 1272..1380 /note="incP origin of transfer" CDS complement(1441..2589) /codon_start=1 /product="trans-acting replication protein that binds to and activates oriV" /label=trfA /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR" rep_origin 2981..3512 /label=oriV /note="incP origin of replication" terminator 3639..3725 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" promoter 3742..3819 /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." CDS 3820..4902 /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 complement(3839..3858) /label=LacI-R /note="LacI, reverse primer" promoter 5134..5163 /label=trc promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 5171..5187 /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)." sig_peptide 5245..5310 /gene="pelB (fragment)" /product="leader peptide for secretion" /label=pelB signal sequence CDS 5356..5445 /codon_start=1 /product="two Strep-Tag(R)II moieties connected by a linker for enhanced binding to Strep-Tactin(R), an engineered form of streptavidin (Schmidt et al., 2013)" /label=Twin-Strep-tag /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK" ORIGIN 1 cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga 61 acgctcggtt gccgccgggc gttttttatt ggtgagaatc caggggtccc caataattac 121 gatttacgta tttaaatgaa ccttgaccga acgcagcggt ggtaacggcg cagtggcggt 181 tttcatggct tgttatgact gtttttttgg ggtacagtct atgcctcggg catccaagca 241 gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca 301 gcagggcagt cgccctaaaa caaagttaaa catcatgagg gaagcggtga tcgccgaagt 361 atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct 421 ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca gtgatattga 481 tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga 541 ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac 601 cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt 661 tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat 721 tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc 781 ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac 841 cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac 901 gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc 961 cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca 1021 ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt 1081 tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc aaataagaca agggtccttt 1141 tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt 1201 ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa 1261 cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc 1321 actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg 1381 taggccggcc gcgatgcagg tggctgctga acccccagcc ggaactgacc ccacaaggcc 1441 ctagcgtttg caatgcacca ggtcatcatt gacccaggcg tgttccacca ggccgctgcc 1501 tcgcaactct tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc gggtggaatc 1561 cgatccgcac atgaggcgga aggtttccag cttgagcggg tacggctccc ggtgcgagct 1621 gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg cggtacttct cccatatgaa 1681 tttcgtgtag tggtcgccag caaacagcac gacgatttcc tcgtcgatca ggacctggca 1741 acgggacgtt ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg acaccgattc 1801 caggtgccca acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc gcgacaggca 1861 ttcctcggcc ttcgtgtaat accggccatt gatcgaccag cccaggtcct ggcaaagctc 1921 gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact ccaacacctg 1981 ctgccacacc agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg tgatcttcac 2041 gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga ttttcttgtt 2101 gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc atcgctcgca tcgtgtccgg 2161 ccacggcgca atatcgaaca aggaaagctg catttccttg atctgctgct tcgtgtgttt 2221 cagcaacgcg gcctgcttgg cttcgctgac ctgttttgcc aggtcctcgc cggcggtttt 2281 tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg ccaaacctgc 2341 cgcctcctgt tcgagacgac gcgaacgctc cacggcggcc gatggcgcgg gcagggcagg 2401 gggagccagt tgcacgctgt cgcgctcgat cttggccgta gcttgctgga ctatcgagcc 2461 gacggactgg aaggtttcgc ggggcgcacg catgacggtg cggcttgcga tggtttcggc 2521 atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc ggtcaaacgt 2581 ccgattcatt caccctcctt gcgggattgc cccggaatta attccccgga tcgatccgtc 2641 gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg 2701 cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc 2761 cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc 2821 tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag 2881 caccgtttct gcggactggc tttctacgtg gctgccattt ttggggtgag gccgttcgcg 2941 gccgaggggc gcagcccctg gggggatggg gtgccgcgtt agcgggccgg gagggttcga 3001 gaaggggggg cacccccctt cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta 3061 aaaacaaggt ttataaatat tggtttaaaa gcaggttaaa agacaggtta gcggtggccg 3121 aaaaacgggc ggaaaccctt gcaaatgctg gattttctgc ctgtggacag cccctcaaat 3181 gtcaataggt gcgcccctca tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc 3241 cctcatctgt cagtagtcgc gcccctcaag tgtcaatacc gcagggcact tatccccagg 3301 cttgtccaca tcatctgtgg gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag 3361 gctggccagc tccacgtcgc cggccgaaat cgagcctgcc cctcatctgt caacgccgcg 3421 ccgggtgagt cggcccctca agtgtcaacg tccgcccctc atctgtcagt gagggccaag 3481 ttttccgcga ggtatccaca acgccggcgg ccctacatgg ctctgctgta gtgagtgggt 3541 tgcgctccgg cagcggtcct gatcccccgc agaaaaaaag gatctcaaga agatcctttg 3601 atcttttcta cggcgcgccc agctgtctag ggcggcggat ttgtcctact caggagagcg 3661 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt 3721 atttgatgcc tttaattaat tgacaccatc gaatggtgca aaacctttcg cggtatggca 3781 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac 3841 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc 3901 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac 3961 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc 4021 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc 4081 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt 4141 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg 4201 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt 4261 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg 4321 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc 4381 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc 4441 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa 4501 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat 4561 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat 4621 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc 4681 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc 4741 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa 4801 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 4861 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt 4921 aagttagcgc gaattgatct ggtttgacag cttatcatcg actgcacggt gcaccaatgc 4981 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc 5041 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca 5101 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata 5161 atgtgtggaa ttgtgagcgg ataacaattt cacaccctag gccgcggccg cgcgaattgt 5221 taactttaag aaggagatat acatatgaag tacctgctgc cgaccgccgc cgccggcctg 5281 ctgctgctgg ccgcccagcc ggccatggcc gatccgaatt cgagctccgt cgactctaga 5341 gcggccgcac tcgagagcgc ttggagccac ccgcagttcg agaaaggtgg aggttccgga 5401 ggtggatcgg gaggttcggc gtggagccac ccgcagttcg aaaaataatg atgaaagctt 5461 gcggccgcgt cgtgactggg aaaaccctgg cgactagt //