LOCUS Exported 8064 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS LTRi SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8064) AUTHORS Deans TL, Cantor CR, Collins JJ TITLE A tunable genetic switch based on RNAi and repressor proteins for regulating gene expression in mammalian cells. JOURNAL Cell. 2007 Jul 27. 130(2):363-72. PUBMED 17662949 REFERENCE 2 (bases 1 to 8064) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8064 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 379..395 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" enhancer 462..765 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 766..969 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" CDS 1022..2101 /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(1041..1060) /label=LacI-R /note="LacI, reverse primer" CDS 2117..2137 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" intron 2300..2365 /label=small t intron /note="SV40 (simian virus 40) small t antigen intron" polyA_signal 2525..2646 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2679..2695) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2703..2719 /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)." promoter complement(2727..2757) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2772..2793 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind 3028..3044 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 3051..3069 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 3164..3182 /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O1 for the tetR and tetA genes" primer_bind complement(3194..3213) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(3364..3384) /label=hU6-F /note="Human U6 promoter, forward primer" promoter complement(3530..3548) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3531..3548) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3566..3582) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 3590..3606 /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)." promoter complement(3614..3644) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3659..3680 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3797..3814) /label=L4440 /note="L4440 vector, forward primer" primer_bind 4022..4038 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" enhancer 4105..4408 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 4409..4612 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" protein_bind 4730..4749 /label=lac operator (symmetric) /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). The symmetric lac operator was optimized for tight binding of lac repressor." protein_bind 4773..4792 /label=lac operator (symmetric) /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). The symmetric lac operator was optimized for tight binding of lac repressor." protein_bind 4816..4835 /label=lac operator (symmetric) /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). The symmetric lac operator was optimized for tight binding of lac repressor." primer_bind 5279..5298 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" promoter 5326..5344 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 5355..5972 /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG" primer_bind complement(5428..5447) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" polyA_signal 6017..6138 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" rep_origin complement(6245..6833) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6325..6344) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7004..7864) /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 7627..7646 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7865..7969) /gene="bla" /label=AmpR promoter primer_bind 8037..8055 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ctagttatta atagtaatca 421 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta 481 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat 541 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg 601 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac 661 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt 721 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg 781 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 841 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 901 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 961 agcagagctg gtttagtgaa ccgtcagatc ggatcctcta gagtcgacca ttgccgccac 1021 catgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt 1081 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc 1141 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca 1201 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt 1261 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga 1321 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag 1381 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg 1441 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat 1501 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca 1561 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg 1621 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg 1681 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac 1741 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc 1801 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc 1861 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag 1921 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc 1981 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg 2041 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 2101 gagcagcctg aggcctccta agaagaagag gaaggtttga gcgcaacgca attaatgtaa 2161 gttagctcac tcattaggca ccccaggctt tacactttat gcttccgacc tgcagcccct 2221 tgggatcttt gtgaaggaac cttacttctg tggtgtgaca taattggaca aactacctac 2281 agagatttaa agctctaagg taaatataaa atttttaagt gtataatgtg ttaaactact 2341 gattctaatt gtttgtgtat tttagattca cagtcccaag gctcatttca ggcccctcag 2401 tcctcacagt ctgttcatga tcataatcag ccataccaca tttgtagagg ttttacttgc 2461 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 2521 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 2581 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 2641 atcttatcat gtctgggccc aagcttggcg taatcatggt catagctgtt tcctgtgtga 2701 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 2761 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 2821 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 2881 ggtttgcgta ttgggcgctc ttccgctgca actgttggga agggcgatcg gtgcgggcct 2941 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 3001 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact 3061 cactataggg cgaattgggc ctttttctac acaaatcagc gattttctct tgaaaaatcg 3121 ctgatttgtg tagcggtgtt tcgtcctttc cacaagatat ataactctat caatgataga 3181 gtactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc 3241 aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt 3301 tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat gcaaatatga 3361 aggaatcatg ggaaataggc cctcttcctg cccgaccttg gcgcgcgctc ggcgcgcggt 3421 cacgctccgt cacgtggtgc gttttgccta atcactagtg aattcgcggc cgcctgcagg 3481 tcgaccatat gggagagctc ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc 3541 acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 3601 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 3661 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 3721 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 3781 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 3841 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 3901 gaaagaacat ggccggctta atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 3961 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 4021 tgtaaaacga cggccagtga attctagtta ttaatagtaa tcaattacgg ggtcattagt 4081 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 4141 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 4201 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 4261 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 4321 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 4381 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 4441 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 4501 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 4561 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt 4621 gaaccgtcag atcggatcct ctagagtcga cctgcaggat ctaagcttgg acaaactacc 4681 tacagagatt taaagctcta aggtaaatat aaaatttact aggttgtgga attgtgagcg 4741 ctcacaattc cacagtcgac cctaggttgt ggaattgtga gcgctcacaa ttccacagtc 4801 gaccctaggt tgtggaattg tgagcgctca caattccaca gtcgacccta gtgtataatg 4861 tgttaaacta cggatccgtc tcccattagg cctacaatgg tgagacaagt agccaacagg 4921 gaagggttgc aaatatcatt tgggcacacc tatgataata ttgatgaagc agacagtatt 4981 cagcaagtaa ctgagaggtg ggaagctcaa agccaaagtc ctaatgtgca gtcaggtgaa 5041 tttattgaaa aatttgaggc tcctggtggt gcaaatcaaa gaactgctcc tcagggatcc 5101 taattgtttg tgtattttag attccaacca agcttgcggc cgctcaggag ctaaggaagc 5161 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa 5221 agaacgggcc cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 5281 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattgtaata cgactcacta 5341 tagggcgaat tgatatgtct agattagata aaagtaaagt gattaacagc gcattagagc 5401 tgcttaatga ggtcggaatc gaaggtttaa caacccgtaa actcgcccag aagctaggtg 5461 tagagcagcc tacattgtat tggcatgtaa aaaataagcg ggctttgctc gacgccttag 5521 ccattgagat gttagatagg caccatactc acttttgccc tttagaaggg gaaagctggc 5581 aagatttttt acgtaataac gctaaaagtt ttagatgtgc tttactaagt catcgcgatg 5641 gagcaaaagt acatttaggt acacggccta cagaaaaaca gtatgaaact ctcgaaaatc 5701 aattagcctt tttatgccaa caaggttttt cactagagaa tgcattatat gcactcagcg 5761 ctgtggggca ttttacttta ggttgcgtat tggaagatca agagcatcaa gtcgctaaag 5821 aagaaaggga aacacctact actgatagta tgccgccatt attacgacaa gctatcgaat 5881 tatttgatca ccaaggtgca gagccagcct tcttattcgg ccttgaattg atcatatgcg 5941 gattagaaaa acaacttaaa tgtgaaagtg ggtccgcgta cagcggatcc cgggaattca 6001 gatcttatta aagcagaact tgtttattgc agcttataat ggttacaaat aaagcaatag 6061 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 6121 actcatcaat gtatcttatc atgtctggtc gaatccatca cactggcggc cgctcgagac 6181 atgtcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 6241 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 6301 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 6361 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 6421 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 6481 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 6541 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 6601 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 6661 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 6721 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 6781 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 6841 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6901 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6961 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 7021 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 7081 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 7141 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 7201 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 7261 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 7321 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 7381 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 7441 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 7501 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 7561 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 7621 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 7681 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 7741 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 7801 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 7861 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 7921 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 7981 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 8041 ggcgtatcac gaggcccttt cgtc //