LOCUS Exported 6585 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION E.coli entry and expression vector for FX cloning system, N-terminal pelB signal sequence and C-terminal fusion to PIII for phage display using M13 phages. ACCESSION . VERSION . KEYWORDS pDXinit SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6585) AUTHORS Zimmermann I, Egloff P, Hutter CA, Arnold FM, Stohler P, Bocquet N, Hug MN, Huber S, Siegrist M, Hetemann L, Gera J, Gmur S, Spies P, Gygax D, Geertsma ER, Dawson RJ, Seeger MA TITLE Synthetic single domain antibodies for the conformational trapping of membrane proteins. JOURNAL Elife. 2018 May 24;7. pii: 34317. doi: 10.7554/eLife.34317. PUBMED 29792401 REFERENCE 2 (bases 1 to 6585) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6585 /organism="synthetic DNA construct" /mol_type="other DNA" sig_peptide 21..86 /gene="pelB (fragment)" /product="leader peptide for secretion" /label=pelB signal sequence promoter 107..552 /gene="sacR" /label=sacB promoter /note="sacB promoter and control region" CDS 553..1974 /codon_start=1 /gene="Bacillus subtilis sacB" /product="secreted levansucrase that renders bacterial growth sensitive to sucrose" /label=SacB /note="negative selection marker" /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV VKDSILEQGQLTVNK" terminator 2048..2134 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" CDS 2208..2225 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3510..3527) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3510..3526) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3519..3541) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3739..4194 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3826..3845) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4036..4057 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4211..4230) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4330..4352 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4390..4408) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4476..4580 /gene="bla" /label=AmpR promoter CDS 4581..5441 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(4799..4818) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5612..6200 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6101..6120 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6354..6371 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6488..6509 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6524..6554 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6562..6578 /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)." ORIGIN 1 agcttaagga gacagtacat atgaaatacc tattgcctac ggcagccgct ggattgttat 61 tactcgcggc ccagccggcc atggcagccg gctcttcaag tctgcccaca tatacctgcc 121 gttcactatt atttagtgaa atgagatatt atgatatttt ctgaattgtg attaaaaagg 181 caactttatg cccatgcaac agaaactata aaaaatacag agaatgaaaa gaaacagata 241 gattttttag ttctttaggc ccgtagtctg caaatccttt tatgattttc tatcaaacaa 301 aagaggaaaa tagaccagtt gcaatccaaa cgagagtcta atagaatgag gtcgaaaagt 361 aaatcgcgcg ggtttgttac tgataaagca ggcaagacct aaaatgtgta aagggcaaag 421 tgtatacttt ggcgtcaccc cttacatatt ttaggtcttt ttttattgtg cgtaactaac 481 ttgccatctt caaacaggag ggctggaaga agcagaccgc taacacagta cataaaaaag 541 gagacatgaa cgatgaacat caaaaagttt gcaaaacaag caacagtatt aacctttact 601 accgcactgc tggcaggagg cgcaactcaa gcgtttgcga aagaaacgaa ccaaaagcca 661 tataaggaaa catacggcat ttcccatatt acacgccatg atatgctgca aatccctgaa 721 cagcaaaaaa atgaaaaata taaagttcct gagttcgatt cgtccacaat taaaaatatc 781 tcttctgcaa aaggcctgga cgtttgggac agctggccat tacaaaacac tgacggcact 841 gtcgcaaact atcacggcta ccacatcgtc tttgcattag ccggagatcc taaaaatgcg 901 gatgacacat cgatttacat gttctatcaa aaagtcggcg aaacttctat tgacagctgg 961 aaaaacgctg gccgcgtctt taaagacagc gacaaattcg atgcaaatga ttctatccta 1021 aaagaccaaa cacaagaatg gtcaggttca gccacattta catctgacgg aaaaatccgt 1081 ttattctaca ctgatttctc cggtaaacat tacggcaaac aaacactgac aactgcacaa 1141 gttaacgtat cagcatcaga cagctctttg aacatcaacg gtgtagagga ttataaatca 1201 atctttgacg gtgacggaaa aacgtatcaa aatgtacagc agttcatcga tgaaggcaac 1261 tacagctcag gcgacaacca tacgctgaga gatcctcact acgtagaaga taaaggccac 1321 aaatacttag tatttgaagc aaacactgga actgaagatg gctaccaagg cgaagaatct 1381 ttatttaaca aagcatacta tggcaaaagc acatcattct tccgtcaaga aagtcaaaaa 1441 cttctgcaaa gcgataaaaa acgcacggct gagttagcaa acggcgctct cggtatgatt 1501 gagctaaacg atgattacac actgaaaaaa gtgatgaaac cgctgattgc atctaacaca 1561 gtaacagatg aaattgaacg cgcgaacgtc tttaaaatga acggcaaatg gtacctgttc 1621 actgactccc gcggatcaaa aatgacgatt gacggcatta cgtctaacga tatttacatg 1681 cttggttatg tttctaattc tttaactggc ccatacaagc cgctgaacaa aactggcctt 1741 gtgttaaaaa tggatcttga tcctaacgat gtaaccttta cttactcaca cttcgctgta 1801 cctcaagcga aaggaaacaa tgtcgtgatt acaagctata tgacaaacag aggattctac 1861 gcagacaaac aatcaacgtt tgcgcctagc ttcctgctga acatcaaagg caagaaaaca 1921 tctgttgtca aagacagcat ccttgaacaa ggacaattaa cagttaacaa ataaaaacgc 1981 aaaagaaaat gccgatatcc tattggcatt gcggccgcgg tctcggcggt gggtcgtcta 2041 gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg 2101 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ccctagacct agcggccggc 2161 cgcaagcgct gcacggcctc acgtgacacg tgtctgcagg aagagctcac caccatcacc 2221 atcacgaacc tgaagcccag tacccgtacg acgttccgga ctacggttcc actgttgaaa 2281 gttgtttagc aaaacctcat acagaaaatt catttactaa cgtctggaaa gacgacaaaa 2341 ctttagatcg ttacgctaac tatgagggct gtctgtggaa tgctacaggc gttgtcgttt 2401 gtactggtga cgaaactcag tgttacggta catgggttcc tattgggctt gctatccctg 2461 aaaatgaggg tggtggctct gagggtggcg gttctgaggg tggcggttct gagggtggcg 2521 gtactaaacc tcctgagtac ggtgatacac ctattccggg ctatacttat atcaaccctc 2581 tcgacggcac ttatccgcct ggtactgagc aaaaccccgc taatcctaat ccttctcttg 2641 aggagtctca gcctcttaat actttcatgt ttcagaataa taggttccga aataggcagg 2701 gtgcattaac tgtttatacg ggcactgtta ctcaaggcac tgaccccgtt aaaacttatt 2761 accagtacac tcctgtatca tcaaaagcca tgtatgacgc ttactggaac ggtaaattca 2821 gagactgcgc tttccattct ggctttaatg aggatccatt cgtttgtgaa tatcaaggcc 2881 aatcgtctga cctgcctcaa cctcctgtca atgctggcgg cggctctggt ggtggttctg 2941 gtggcggctc tgagggtggc ggctctgagg gtggcggctc tgagggtggc ggttctgagg 3001 gtggcggctc tgagggtggc ggttccggtg gcggctccgg ttccggtgat tttgattatg 3061 aaaaaatggc aaacgctaat aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac 3121 agtctgacgc taaaggcaaa cttgattctg tcgctactga ttacggtgct gctatcgatg 3181 gtttcattgg tgacgtttcc ggccttgcta atggtaatgg tgctactggt gattttgctg 3241 gctctaattc ccaaatggct caagtcggtg acggtgataa ttcaccttta atgaataatt 3301 tccgtcaata tttaccttct ttacctcagt cggttgaatg tcgcccttat gtctttggcg 3361 ctggtaaacc atatgaattt tctattgatt gtgacaaaat aaacttattc cgtggtgtct 3421 ttgcgtttct tttatatgtt gccaccttta tgtatgtatt ttcgacgttt gctaacatac 3481 tgcgtaataa ggagtcttaa taagaattca ctggccgtcg ttttacaacg tcgtgactgg 3541 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 3601 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 3661 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 3721 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 3781 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct 3841 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 3901 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg 3961 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 4021 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg 4081 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc 4141 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt 4201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 4261 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccacttac agacaagctg 4321 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 4381 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 4441 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 4501 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 4561 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 4621 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 4681 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 4741 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 4801 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 4861 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 4921 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 4981 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 5041 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 5101 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 5161 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 5221 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 5281 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 5341 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 5401 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 5461 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 5521 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 5581 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 5641 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 5701 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 5761 ttctagtgta gccgtagtta ggccactact tcaagaactc tgtagcaccg cctacatacc 5821 tcgcactgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 5881 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 5941 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 6001 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 6061 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 6121 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 6181 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 6241 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 6301 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 6361 cagtgagcga ggaagcggaa gagagcccaa tacgcaaacc gcctctcccc gcgcgttggc 6421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 6481 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 6541 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcaca //