LOCUS Exported 8203 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pRK7-FLAG-TSC1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8203) AUTHORS Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC, Blenis J TITLE Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. JOURNAL Proc Natl Acad Sci U S A. 2002 Oct 15. 99(21):13571-6. PUBMED 12271141 REFERENCE 2 (bases 1 to 8203) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8203 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 14..393 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 394..597 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 548..568 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 594..618 /label=LNCX /note="Human CMV promoter, forward primer" promoter 828..846 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 828..845 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 859..878 /label=pMT2-F /note="Synthetic intron, forward primer" CDS 933..956 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 4455..4464 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 4468..4602 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4505..4524) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4559..4578 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 4675..5032 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(4698..4718) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 4883..5018 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4945..4964 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(5052..5069) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5052..5068) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5061..5083) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5281..5736 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5368..5387) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5578..5599 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5753..5772) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5907..5929 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5982..6000) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6068..6172 /gene="bla" /label=AmpR promoter CDS 6173..7033 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYM ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRDDAGQEQLGRRIHYSQNDLVEYS PATEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(6391..6410) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7204..7792 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7693..7712 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7946..7963 /label=L4440 /note="L4440 vector, forward primer" promoter 8116..8146 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8154..8170 /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 8159..8181 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 8178..8194 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 8178..8194 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca 61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta 781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac 841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc 901 acctcggttc taagcttctg caggtcgaca tggactataa ggacgatgat gacaaagccc 961 aacaagcaaa tgtcggggag cttcttgcca tgctggactc ccccatgctg ggtgtgcggg 1021 acgacgtgac agctgtcttt aaagagaacc tcaattctga ccgtggccct atgcttgtaa 1081 acaccttggt ggattattac ctggaaacca gctctcagcc ggcattgcac atcctgacca 1141 ccttgcaaga gccacatgac aagcacctct tggacaggat taacgaatat gtgggcaaag 1201 ccgccactcg tttatccatc ctctcgttac tgggtcatgt cataagactg cagccatctt 1261 ggaagcataa gctctctcaa gcacctcttt tgccttcttt actaaaatgt ctcaagatgg 1321 acactgacgt cgttgtcctc acaacaggcg tcttggtgtt gataaccatg ctaccaatga 1381 ttccacagtc tgggaaacag catcttcttg atttctttga catttttggc cgtctgtcat 1441 catggtgcct gaagaaaccg ggccacgtgg cggaagtcta tcttgtccat ctccatgcca 1501 gtgtgtacgc actctttcat cgcctttatg gaatgtaccc ttgcaacttc gtctcctttt 1561 tgcgttctca ttacagtatg aaagaaaacc tggagacttt tgaagaagtg gtcaagccaa 1621 tgatggagca tgtgcgaatt catccggaat tagtgactgg atccaaggac catgagctgg 1681 accctcgaag gtggaagaga ttagaaactc atgatgttgt gatcgagtgt gccaaaatct 1741 ctctggatcc cacagaagcc tcatatgaag atggctattc tgtgtctcac caaatctcag 1801 cccgctttcc tcatcgttca gccgatgtca ccaccagccc ttatgctgac acacagaata 1861 gctatgggtg tgctacttct accccttact ccacgtctcg gctgatgttg ttaaatatgc 1921 cagggcagct acctcagact ctgagttccc catcgacacg gctgataact gaaccaccac 1981 aagctactct ttggagccca tctatggttt gtggtatgac cactcctcca acttctcctg 2041 gaaatgtccc acctgatctg tcacaccctt acagtaaagt ctttggtaca actgcaggtg 2101 gaaaaggaac tcctctggga accccagcaa cctctcctcc tccagcccca ctctgtcatt 2161 cggatgacta cgtgcacatt tcactccccc aggccacagt cacacccccc aggaaggaag 2221 agagaatgga ttctgcaaga ccatgtctac acagacaaca ccatcttctg aatgacagag 2281 gatcagaaga gccacctggc agcaaaggtt ctgtcactct aagtgatctt ccagggtttt 2341 taggtgatct ggcctctgaa gaagatagta ttgaaaaaga taaagaagaa gctgcaatat 2401 ctagagaact ttctgagatc accacagcag aggcagagcc tgtggttcct cgaggaggct 2461 ttgactctcc cttttaccga gacagtctcc caggttctca gcggaagacc cactcggcag 2521 cctccagttc tcagggcgcc agcgtgaacc ctgagccttt acactcctcc ctggacaagc 2581 ttgggcctga cacaccaaag caagccttta ctcccataga cctgccctgc ggcagtgctg 2641 atgaaagccc tgcgggagac agggaatgcc agacttcttt ggagaccagt atcttcactc 2701 ccagtccttg taaaattcca cctccgacga gagtgggctt tggaagcggg cagcctcccc 2761 cgtatgatca tctttttgag gtggcattgc caaagacagc ccatcatttt gtcatcagga 2821 agactgagga gctgttaaag aaagcaaaag gaaacacaga ggaagatggt gtgccctcta 2881 cctccccaat ggaagtgctg gacagactga tacagcaggg agcagacgcg cacagcaagg 2941 agctgaacaa gttgccttta cccagcaagt ctgtcgactg gacccacttt ggaggctctc 3001 ctccttcaga tgagatccgc accctccgag accagttgct tttactgcac aaccagttac 3061 tctatgagcg ttttaagagg cagcagcatg ccctccggaa caggcggctc ctccgcaagg 3121 tgatcaaagc agcagctctg gaggaacata atgctgccat gaaagatcag ttgaagttac 3181 aagagaagga catccagatg tggaaggtta gtctgcagaa agaacaagct agatacaatc 3241 agctccagga gcagcatgac actatggtaa ccaagctcca cagccagatc agacagctgc 3301 agcatgaccg agaggaattc tacaaccaga gccaggaatt acagacgaag ctggaggact 3361 gcaggaacat gattgcggag ctgcggatgg aactgaagaa ggccaacaac aaggtgtgtc 3421 acactgagct gctgctcagt caggtttccc aaaagctctc aaacagtgag tcggtccagc 3481 agcagatgga gttcttgaac aggcagctgt tggttcttgg ggaggtcaac gagctctatt 3541 tggaacaact gcagaacaag cactcagata ccacaaagga agtagaaatg atgaaagccg 3601 cctatcggaa agagctagaa aaaaacagaa gccatgttct ccagcagact cagaggcttg 3661 atacctccca aaaacggatt ttggaactgg aatctcacct ggccaagaaa gaccaccttc 3721 ttttggaaca gaagaaatat ctagaggatg tcaaactcca ggcaagagga cagctgcagg 3781 ccgcagagag caggtatgag gctcagaaaa ggataaccca ggtgtttgaa ttggagatct 3841 tagatttata tggcaggttg gagaaagatg gcctcctgaa aaaacttgaa gaagaaaaag 3901 cagaagcagc tgaagcagca gaagaaaggc ttgactgttg taatgacggg tgctcagatt 3961 ccatggtagg gcacaatgaa gaggcatctg gccacaacgg tgagaccaag acccccaggc 4021 ccagcagcgc ccggggcagt agtggaagca gaggtggtgg aggcagcagc agcagcagca 4081 gcgagctttc taccccagag aaacccccac accagagggc aggcccattc agcagtcggt 4141 gggagacgac tatgggagaa gcgtctgcca gcatccccac cactgtgggc tcacttccca 4201 gttcaaaaag cttcctgggt atgaaggctc gagagttatt tcgtaataag agcgagagcc 4261 agtgtgatga ggacggcatg accagtagcc tttctgagag cctaaagaca gaactgggca 4321 aagacttggg tgtggaagcc aagattcccc tgaacctaga tggccctcac ccgtctcccc 4381 cgaccccgga cagtgttgga cagctacata tcatggacta caatgagact catcatgaac 4441 acagctaatc gatggccgcc atggcccaac ttgtttattg cagcttataa tggttacaaa 4501 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 4561 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcgatcggga attaattcgg 4621 cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta ccttctgagg 4681 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 4741 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 4801 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 4861 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 4921 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctgtga 4981 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctgtt 5041 aacagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 5101 caacttaatc gccttgcagc acatcccccc ttcgccacct ggcgtaatag cgaagaggcc 5161 cgcaccgatc gcccttccca acagttgcgt agcctgaatg gcgaatggcg cctgatgcgg 5221 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt 5281 acgcgccctg tacgggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 5341 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 5401 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 5461 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc 5521 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 5581 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat 5641 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 5701 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct 5761 gctctgatgc cgcatagtta agccaactcc gctatcgcta cgtgactggg tcatggctgc 5821 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 5881 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 5941 atcaccgaaa cgcgcgaggc agtattcttg aagacgaaag ggcctcgtga tacgcctatt 6001 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 6061 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 6121 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 6181 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 6241 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 6301 ttacatggaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 6361 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgatga 6421 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 6481 ctcaccagcc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 6541 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 6601 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 6661 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgccagcagc 6721 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 6781 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 6841 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 6901 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 6961 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 7021 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 7081 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 7141 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 7201 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 7261 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 7321 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 7381 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 7441 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 7501 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 7561 gacctacacc gaactgagat acctacagcg tgagcattga gaaagcgcca cgcttcccga 7621 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 7681 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 7741 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 7801 caacgcggcc tttttacggt tcctggcctt ttggtggcct tttgctcaca tgttctttcc 7861 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 7921 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 7981 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatccagct ggcacgacag 8041 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca 8101 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 8161 cggataacaa tttcacacag gaaacagcta tgaccatgat tac //