LOCUS Exported 5265 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pRK5-myc-Cdc42-Q61L SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5265) TITLE Bokoch Lab GTPases REFERENCE 2 (bases 1 to 5265) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5265 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /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 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" enhancer 250..629 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 630..833 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 784..804 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 830..854 /label=LNCX /note="Human CMV promoter, forward primer" promoter 1064..1082 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 1064..1081 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 1095..1114 /label=pMT2-F /note="Synthetic intron, forward primer" regulatory 1161..1170 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1170..1199 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 1206..1781 /codon_start=1 /product="constitutively active mutant of human Rho family small GTPase Cdc42" /label=Cdc42 (Q61L) /translation="MQTIKCVVVGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVM IGGEPYTLGLFDTAGLEDYDRLRPLSYPQTDVFLVCFSVVSPSSFENVKEKWVPEITHH CPKTPFLLVGTQIDLRDDPSTIEKLAKNKQKPITPETAEKLARDLKAVKYVECSALTQK GLKNVFDEAILAALEPPEPKKSRRCVLL" regulatory 1801..1810 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 1814..1948 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1851..1870) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1905..1924 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 2017..2374 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2040..2060) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2225..2360 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2287..2306 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2394..2411) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2394..2410) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2403..2425) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2623..3078 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2710..2729) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2920..2941 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3095..3114) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3214..3236 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3274..3292) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3360..3464 /gene="bla" /label=AmpR promoter CDS 3465..4325 /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 complement(3683..3702) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4496..5084 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4985..5004 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5238..5255 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga catgattacg aattaattcg 241 agctcgcccg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 301 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 361 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 421 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 481 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 541 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 601 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 661 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 721 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 781 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag 841 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc 901 gggaccgatc cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca 961 agagtgacgt aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac 1021 gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg tgacactata 1081 gaataacatc cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct 1141 cggttctatc gattgaattg gccaccatgg agcagaagct gatctccgag gaggacctgg 1201 gatccatgca gacaattaag tgtgttgttg tgggcgatgg tgctgttggt aaaacatgtc 1261 tcctgatatc ctacacaaca aacaaatttc catcggaata tgtaccgact gtttttgaca 1321 actatgcagt cacagttatg attggtggag aaccatatac tcttggactt tttgatactg 1381 cagggctaga ggattatgac agattacgac cgctgagtta tccacaaaca gatgtatttc 1441 tagtctgttt ttcagtggtc tctccatctt catttgaaaa cgtgaaagaa aagtgggtgc 1501 ctgagataac tcaccactgt ccaaagactc ctttcttgct tgttgggact caaattgatc 1561 tcagagatga cccctctact attgagaaac ttgccaagaa caaacagaag cctatcactc 1621 cagagactgc tgaaaagctg gcccgtgacc tgaaggctgt caagtatgtg gagtgttctg 1681 cacttacaca gaaaggccta aagaatgtat ttgacgaagc aatattggct gccctggagc 1741 ctccagaacc gaagaagagc cgcaggtgtg tgctgctatg agaattcctg cagaagcttg 1801 gccgccatgg cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 1861 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 1921 catcaatgta tcttatcatg tctggatcgg gaattaattc ggcgcagcac catggcctga 1981 aataacctct gaaagaggaa cttggttagg taccttctga ggcggaaaga accagctgtg 2041 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 2101 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg 2161 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc 2221 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 2281 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg 2341 aggaggcttt tttggaggcc taggcttttg caaaaagctg ttaacagctt ggcactggcc 2401 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 2461 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 2521 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat 2581 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg 2641 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2701 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 2761 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 2821 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 2881 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 2941 gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt 3001 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 3061 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt 3121 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 3181 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 3241 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 3301 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 3361 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 3421 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 3481 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 3541 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 3601 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 3661 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 3721 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 3781 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 3841 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 3901 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 3961 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 4021 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 4081 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 4141 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 4201 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 4261 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 4321 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 4381 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 4441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 4501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 4561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 4621 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 4681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 4741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 4801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 4861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 4921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 4981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 5041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 5101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 5161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 5221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag //