LOCUS Exported 6025 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pET PPL His6 MBP LIC cloning vector (2K-T) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6025) TITLE 2-series LIC periplasmic secretion vectors for E. coli JOURNAL Unpublished REFERENCE 2 (bases 1 to 6025) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6025 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(18..36) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 104..208 /gene="bla" /label=AmpR promoter CDS 209..1069 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(427..446) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1240..1828 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1729..1748 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1982..1999 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2014..2156 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2082..2101) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2242..2264 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2258..2449) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind 3907..3926 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4010..4029 /label=T7 /note="T7 promoter, forward primer" promoter 4010..4028 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 4117..4139 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 4234..4251 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 4261..5361 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" primer_bind 5335..5358 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 5416..5436 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" primer_bind complement(5561..5579) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5575..5622 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(5988..6016) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" ORIGIN 1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc 3721 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc 3781 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc 3841 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc 3901 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc 3961 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc 4021 actataggga gaccacaacg gtttccctct agtgccggct ccggagagct ctttaattaa 4081 gcggccgccc tgcaggactc gagttctaga aataattttg tttaacttta agaaggagat 4141 atacatatga aaataaaaac aggtgcacgc atcctcgcat tatccgcatt aacgacgatg 4201 atgttttccg cctcggctct cgccaaatct tctcaccatc accatcacca tggttcttct 4261 atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 4321 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 4381 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 4441 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 4501 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 4561 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 4621 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 4681 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 4741 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 4801 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 4861 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 4921 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 4981 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 5041 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 5101 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 5161 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 5221 actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 5281 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 5341 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 5401 aacctcggga tcgaggaaaa cctgtacttc caatccaata ttggaagtgg ataacggatc 5461 cgcgatcgcg gcgcgccacc tggtggccgg ccggtaccac gcgtgcgcgc tgatccggct 5521 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca 5581 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata 5641 tccggatatc cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag tggctccaag 5701 tagcgaagcg agcaggactg ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg 5761 tgcgcataga aattgcatca acgcatatag cgctagcagc acgccatagt gactggcgat 5821 gctgtcggaa tggacgatat cccgcaagag gcccggcagt accggcataa ccaagcctat 5881 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat 5941 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc 6001 gatgataagc tgtcaaacat gagaa //