LOCUS Exported 6148 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Used for tac promoter regulated high-level expression of cloned, untagged genes in E. Coli.. ACCESSION . VERSION . KEYWORDS pTRC-LIC SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6148) TITLE Arrowsmith Lab Plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 6148) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6148 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..92 /gene="bla" /label=AmpR promoter CDS 93..953 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPTAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(311..330) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1124..1712 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1613..1632 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1866..1883 /label=L4440 /note="L4440 vector, forward primer" misc_feature 1898..2038 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(1964..1983) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2124..2146 /label=pGEX 3' /note="pGEX vectors, reverse primer" promoter 2224..2301 /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 2302..3384 /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 YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(2321..2340) /label=LacI-R /note="LacI, reverse primer" promoter 3616..3645 /label=trc promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 3653..3669 /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)." primer_bind 3658..3680 /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3720..3753 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 3768..4213 /gene="sacR" /label=sacB promoter /note="sacB promoter and control region" CDS 4214..5635 /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(5754..5771) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 5924..6010 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 6102..6129 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" ORIGIN 1 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 61 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 121 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 181 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 241 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 301 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg 361 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 421 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 481 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 541 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 601 ccataccaaa cgacgagcgt gacaccacga tgcctacagc aatggcaaca acgttgcgca 661 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 721 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 781 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 841 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 901 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 961 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 1021 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 1081 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 1141 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 1201 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 1261 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 1321 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 1381 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 1441 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 1501 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 1561 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 1621 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 1681 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 1741 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 1801 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 1861 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta 1921 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg 1981 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc 2041 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 2101 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 2161 accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcatttac 2221 gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag 2281 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc 2341 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa 2401 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca 2461 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg 2521 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc 2581 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat 2641 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc 2701 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag 2761 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat 2821 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg 2881 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata 2941 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg 3001 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca 3061 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac 3121 gacgataccg aagacagctc atgttatatc ccgccgtcaa ccaccatcaa acaggatttt 3181 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg 3241 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat 3301 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt 3361 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc gcgaattgat 3421 ctggtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc 3481 atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa 3541 ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata 3601 ttctgaaatg agctgttgac aattaatcat ccggctcgta taatgtgtgg aattgtgagc 3661 ggataacaat ttcacacagg aaacagacca tgattatgag ttctcctcct gaaagatcca 3721 taacttcgta tagcatacat tatacgaagt tatgcggccg cgacgtccac atatacctgc 3781 cgttcactat tatttagtga aatgagatat tatgatattt tctgaattgt gattaaaaag 3841 gcaactttat gcccatgcaa cagaaactat aaaaaataca gagaatgaaa agaaacagat 3901 agatttttta gttctttagg cccgtagtct gcaaatcctt ttatgatttt ctatcaaaca 3961 aaagaggaaa atagaccagt tgcaatccaa acgagagtct aatagaatga ggtcgaaaag 4021 taaatcgcgc gggtttgtta ctgataaagc aggcaagacc taaaatgtgt aaagggcaaa 4081 gtgtatactt tggcgtcacc ccttacatat tttaggtctt tttttattgt gcgtaactaa 4141 cttgccatct tcaaacagga gggctggaag aagcagaccg ctaacacagt acataaaaaa 4201 ggagacatga acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat taacctttac 4261 taccgcactg ctggcaggag gcgcaactca agcgtttgcg aaagaaacga accaaaagcc 4321 atataaggaa acatacggca tttcccatat tacacgccat gatatgctgc aaatccctga 4381 acagcaaaaa aatgaaaaat atcaagttcc tgagttcgat tcgtccacaa ttaaaaatat 4441 ctcttctgca aaaggcctgg acgtttggga cagctggcca ttacaaaacg ctgacggcac 4501 tgtcgcaaac tatcacggct accacatcgt ctttgcatta gccggagatc ctaaaaatgc 4561 ggatgacaca tcgatttaca tgttctatca aaaagtcggc gaaacttcta ttgacagctg 4621 gaaaaacgct ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg attctatcct 4681 aaaagaccaa acacaagaat ggtcaggttc agccacattt acatctgacg gaaaaatccg 4741 tttattctac actgatttct ccggtaaaca ttacggcaaa caaacactga caactgcaca 4801 agttaacgta tcagcatcag acagctcttt gaacatcaac ggtgtagagg attataaatc 4861 aatctttgac ggtgacggaa aaacgtatca aaatgtacag cagttcatcg atgaaggcaa 4921 ctacagctca ggcgacaacc atacgctgag agatcctcac tacgtagaag ataaaggcca 4981 caaatactta gtatttgaag caaacactgg aactgaagat ggctaccaag gcgaagaatc 5041 tttatttaac aaagcatact atggcaaaag cacatcattc ttccgtcaag aaagtcaaaa 5101 acttctgcaa agcgataaaa aacgcacggc tgagttagca aacggcgctc tcggtatgat 5161 tgagctaaac gatgattaca cactgaaaaa agtgatgaaa ccgctgattg catctaacac 5221 agtaacagat gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat ggtacctgtt 5281 cactgactcc cgcggatcaa aaatgacgat tgacggcatt acgtctaacg atatttacat 5341 gcttggttat gtttctaatt ctttaactgg cccatacaag ccgctgaaca aaactggcct 5401 tgtgttaaaa atggatcttg atcctaacga tgtaaccttt acttactcac acttcgctgt 5461 acctcaagcg aaaggaaaca atgtcgtgat tacaagctat atgacaaaca gaggattcta 5521 cgcagacaaa caatcaacgt ttgcgcctag cttcctgctg aacatcaaag gcaagaaaac 5581 atctgttgtc aaagacagca tccttgaaca aggacaatta acagttaaca aataaaaacg 5641 caaaagaaaa tgccgatatc ctattggcat tgacgtcagg tggcactttt cgaggagatc 5701 atgcacatga tgacgaagct tggctgtttt ggcggatgag agaagatttt cagcctgata 5761 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc 5821 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt 5881 agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc 5941 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag 6001 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg 6061 ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga 6121 tggccttttt gcgtttctac aaactctt //