LOCUS Exported 7737 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION SGC Empty backbone for bacterial expression under T7 promoter. Uses infusion based cloning method.. ACCESSION . VERSION . KEYWORDS pET15-MHL SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7737) TITLE Cloning Vectors REFERENCE 2 (bases 1 to 7737) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7737 /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" CDS complement(3761..4843) /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 4805..4824 /label=LacI-R /note="LacI, reverse primer" promoter complement(4844..4921) /gene="lacI" /label=lacI promoter primer_bind 5127..5146 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5230..5249 /label=T7 /note="T7 promoter, forward primer" promoter 5230..5248 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5249..5273 /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 5288..5310 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5320..5337 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5350..5370 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" protein_bind 5398..5431 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 5446..5891 /gene="sacR" /label=sacB promoter /note="sacB promoter and control region" CDS 5892..7313 /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 ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV VKDSILEQGQLTVNK" primer_bind complement(7464..7482) /label=T7 Term /note="T7 terminator, reverse primer" terminator 7478..7525 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(7700..7728) /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 tattgaaaaa 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 gagatcccgg 3721 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 3781 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 3841 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 3901 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 3961 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 4021 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 4081 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 4141 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 4201 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 4261 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 4321 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 4381 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 4441 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 4501 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 4561 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 4621 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 4681 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 4741 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 4801 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 4861 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 4921 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 4981 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 5041 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 5101 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 5161 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 5221 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 5281 aaataatttt gtttaacttt aagaaggaga tataccatgc atcatcatca tcatcacagc 5341 agcggcagag aaaacttgta tttccagggc catatgagtt ctcctcctga aagatccata 5401 acttcgtata gcatacatta tacgaagtta tgcggccgcg acgtccacat atacctgccg 5461 ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc 5521 aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag 5581 attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa 5641 agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta 5701 aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt 5761 gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact 5821 tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg 5881 agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta 5941 ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat 6001 ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac 6061 agcaaaaaaa tgaaaaatat caagttcctg agttcgattc gtccacaatt aaaaatatct 6121 cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg 6181 tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg 6241 atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga 6301 aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa 6361 aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt 6421 tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag 6481 ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa 6541 tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact 6601 acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca 6661 aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt 6721 tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac 6781 ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg 6841 agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag 6901 taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca 6961 ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc 7021 ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg 7081 tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac 7141 ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg 7201 cagacaaaca atcaacgttt gcgcctagct tcctgctgaa catcaaaggc aagaaaacat 7261 ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca 7321 aaagaaaatg ccgatatcct attggcattg acgtcaggtg gcacttttcg aggagatcat 7381 gcacatgatg acgaagcttg cggccgcact cgaggatccg gctgctaaca aagcccgaaa 7441 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc 7501 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat atcccgcaag 7561 aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg 7621 aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt 7681 aactgtgata aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaa //