LOCUS V035294 10806 bp DNA circular SYN 01-JAN-1980 DEFINITION synthetic circular DNA. ACCESSION V035294 VERSION V035294 KEYWORDS . SOURCE . ORGANISM . . FEATURES Location/Qualifiers enhancer 235..614 /note="human cytomegalovirus immediate early enhancer" /label="CMV enhancer" promoter 615..818 /note="human cytomegalovirus (CMV) immediate early promoter" /label="CMV promoter" promoter 863..881 /note="promoter for bacteriophage T7 RNA polymerase" /label="T7 promoter" regulatory 913..922 /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" /regulatory_class="other" /label="Kozak sequence" CDS 922..987 /codon_start=1 /product="three tandem FLAG® epitope tags, followed by an enterokinase cleavage site" /transl_table=1 /translation="DYKDHDGDYKDHDIDYKDDDDK" /label="3xFLAG" CDS 991..5595 /label="Robo2(NM_001358493)" /note="Robo2(NM_001358493)" /gene="Robo2" CDS 5602..5655 /codon_start=1 /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /product="2A peptide from Thosea asigna virus capsid protein" /protein_id="" /transl_table=1 /translation="EGRGSLLTCGDVEENPGP" /label="T2A" CDS 5656..6375 /codon_start=1 /note="mammalian codon-optimized" /product="the original enhanced GFP (Yang et al., 1996)" /transl_table=1 /translation="M,V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV LLEFVTAAGITLGMDELYK*" /label="EGFP" polyA_signal 6406..6630 /note="bovine growth hormone polyadenylation signal" /label="bGH poly(A) signal" rep_origin 6676..7104 /direction=RIGHT /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" /label="f1 ori" promoter 7118..7447 /note="SV40 enhancer and early promoter" /label="SV40 promoter" CDS 7514..8308 /codon_start=1 /gene="aph(3')-II (or nptII)" /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin®)" /product="aminoglycoside phosphotransferase from Tn5" /transl_table=1 /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" /label="NeoR/KanR" polyA_signal 8482..8603 /note="SV40 polyadenylation signal" /label="SV40 poly(A) signal" primer_bind complement(8652..8668) /note="common sequencing primer, one of multiple similar variants" /label="M13 rev" protein_bind 8676..8692 /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-β-D-thiogalactopyranoside (IPTG)." /label="lac operator" promoter complement(8700..8730) /note="promoter for the E. coli lac operon" /label="lac promoter" protein_bind 8745..8766 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." /label="CAP binding site" rep_origin complement(9054..9639) /direction=LEFT /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /label="ori" CDS complement(9810..10670) /codon_start=1 /gene="bla" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /product="β-lactamase" /transl_table=1 /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA SLIKHW*" /label="AmpR" promoter complement(10671..10775) /gene="bla" /label="AmpR promoter" ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 901 gtttaaactt aagccaccat ggactacaaa gaccatgacg gtgattataa agatcatgac 961 atcgactaca aggatgacga tgacaagctt atgttatcat ccgagaaagt ggacaaaatt 1021 actagactcg aacttctctt cgttttgatg ttattcttct caggaccaac tctaggcaag 1081 ctgaccagga ccccctggaa gggcaaacct tggaaggggg gatctcgtct tcgtcaagaa 1141 gactttcccc ccaggatcgt agaacaccct tctgatgtca tcgtctccaa gggagagccc 1201 accactctga actgtaaagc agagggccga cccaccccca ccattgaatg gtacaaggat 1261 ggtgagaggg tggagacaga caaggatgat cccaggtccc acagaatgct tctgcccagc 1321 ggatctttat tctttttgcg aattgttcat gggcgcagaa gtaaaccgga cgaagggagt 1381 tacgtttgtg ttgcaaggaa ctatcttggt gaagcagtga gtcgaaatgc atctctggaa 1441 gtggcattat tgcgagatga cttccggcaa aaccccacag atgtggtagt cgcagctgga 1501 gagcctgcaa tcttggagtg ccagccacca cggggacacc cagaaccaac catctactgg 1561 aaaaaggaca aagtccgaat tgatgacaag gaagagagaa taagtatccg tggtgggaag 1621 ctgatgatct ctaatactag gaaaagcgat gctggcatgt acacctgtgt gggaaccaat 1681 atggtgggag aaagggacag cgaccctgca gagctcactg tctttgaacg acccacattt 1741 ctcaggaggc caattaacca ggtggtgcta gaggaagaag ctgtagaatt ccgttgtcag 1801 gtccaaggag atccccagcc aacggtgagg tggaaaaaag atgatgcaga cttgccgaga 1861 ggaaggtatg atatcaaaga tgactacacg ctgagaatta aaaaggccat gagtactgat 1921 gaaggtacct atgtgtgtat tgctgagaat cgggtgggaa aagtggaagc ctctgctacc 1981 ctcactgtcc gagttcgccc tgttgctcct ccacagtttg tggttaggcc aagagatcag 2041 atcgttgctc aaggccgaac agtgacattc ccctgtgaaa ctaaaggaaa cccacagcca 2101 gctgtttttt ggcagaaaga aggcagccag aacctacttt tcccgaatca acctcagcag 2161 cccaacagcc gatgttcagt gtcgcccacg ggggacctca ccatcaccaa catccagcgt 2221 tcagatgcgg gttactacat ctgccaggcc ctaaccgtgg caggaagcat tttagctaaa 2281 gcacagttgg aagttactga cgttttgaca gatagacctc cacccataat cttgcaagga 2341 ccaataaacc aaacacttgc agtagacggt acagcattgt tgaagtgtaa agccactggt 2401 gagcctctgc ctgtaattag ctggctaaag gagggcttta cttttctggg gagagatcca 2461 agagccacga tccaagacca aggaacactg cagattaaga atttacggat atctgatact 2521 ggcacttata cttgtgtggc tacaagttcc agtggagaga cttcctggag tgcagtgctg 2581 gatgtaacag aatctggagc aacaatcagt aaaaattatg atatgaatga cctcccggga 2641 ccaccatcca aacctcaggt cactgatgtt tctaagaaca gtgtcacctt atcctggcag 2701 ccaggtacac ctggcgttct tcctgcaagc gcgtatatca ttgaggcttt cagccaatcg 2761 gtgagcaata gctggcagac agtggcaaac catgttaaga caactctgta tacagtaagg 2821 gggctgaggc ccaacacaat ctacttgttt atggtcagag cgatcaaccc acaaggtctc 2881 agtgatccaa gtcctatgtc ggatcctgta cgcacacaag atatcagccc cccagcacaa 2941 ggagtggacc acagacaggt gcagaaggaa ttaggtgatg tcgttgttcg tctccataat 3001 ccagttgtcc tgacacctac aactgttcaa gtcacatgga cggtggaccg acaaccccag 3061 tttattcagg gctacagagt gatgtaccgt cagacttcgg gactacaagc ctcaactgtg 3121 tggcagaatc tagacgccaa agtcccgact gagaggagtg ctgtccttgt gaatttgaaa 3181 aagggggtga cttatgaaat taaagtccgg ccgtatttta acgagttcca aggaatggac 3241 agtgaatcga aaacagtccg aaccactgag gaagccccaa gtgcccctcc ccagtctgtc 3301 actgtgctga cagttggaag tcacaacagc acaagcatca gtgtttcctg ggatcctcca 3361 ccagccgacc accagaatgg aattattcag gaatataaga tctggtgtct gggaaacgaa 3421 acgcgattcc atatcaataa aacggtggat gcagccattc gctctgtagt aataggtggc 3481 ttgttccctg gaattcagta ccgggtagaa gtggcagcta gcacaagtgc aggggttgga 3541 gtaaaaagtg aaccacagcc gataataatt gggggacgta acgaagttgt cattactgaa 3601 aacaataaca gcatcactga gcaaatcacg gatgtcgtga agcaaccggc atttatagct 3661 ggcattggtg gtgcctgctg ggtaattctg atgggtttta gcatctggtt gtactggaga 3721 agaaagaaga gaaagggact cagcaattat gctgtaacat ttcaaagagg agatggagga 3781 ctaatgagca atgggagccg tccaggtctt ctaaatgctg gcgatcccaa ttacccatgg 3841 cttgctgatt cttggccagc cacgagtttg ccagtgaaca atagcaatag tggcccaaat 3901 gaaattggaa attttgggcg tggagatgtg ctgcctccgg tgccaggcca aggggataaa 3961 acagcgacca tgctctcgga tggagccatt tatagcagca ttgacttcac taccaaaacc 4021 acttacaaca gttccagcca aataacacag gccaccccat atgccactac acaaatcctg 4081 cattcaaaca gcatccacga actggcagtt gatcttcctg atccacagtg gaaaagctca 4141 gttcaacaga agacagacct catgggattt ggttattcgc tacctgatca gaacaagggg 4201 aacaacgcct tactttacat ccctgactac cgattggctg agggattgtc taatagaatg 4261 ccacacaacc agtcacagga tttcagcacc accagctctc acaacagctc agaaaggagt 4321 ggcagtctct caggtgggaa aggtggaaaa aagaagaaaa ctaaaaattc ttcgaaagcg 4381 cagaaaaaca acggatccac ttgggctaat gtccctctac ctcctcctcc cgtccagccc 4441 cttcctggta cagagctggg ccactatgct gcggaacaag aaaatggcta tgacagtgac 4501 agctggtgcc caccattacc ggtgcaaaca tacttgcatc agggtatgga agatgagctg 4561 gaagaagacg aagatcgggt cccaacacct cctgttcgcg gcgtggcctc ttcaccagct 4621 atctcttttg gacagcagtc cactgccact cttactccat ccccacggga agagatgcaa 4681 cccatgctgc aagctcactt ggatgagttg acaagggcct atcagtttga tatagcaaaa 4741 caaacatggc acattcaaag caatacccca cctccacaac ccccagctcc gccattaggt 4801 tacgtgtccg gagccttgat ttctgatttg gagacagatg ttccagatga ggatgctgat 4861 gatgaagagg aaccattaga aattcccagg cccctcagag cactagacca gacacctgga 4921 tccagtatgg acaatctaga cagctctgtc acaggaaaag cctttagctc ctctcaaagg 4981 cagcggccca ccagcccatt ttctacggac agtaacacca gtgctgccca gaatcaaagc 5041 cagaggcctc ggcccacgaa aaaacataag ggaggacgga tggacccaca gccagtgtta 5101 cctcatcgga gggaagggat gccagatgat cttcccccac caccagatcc cccaccaggt 5161 cagggtttaa ggcagcagat aggcctgagc caacactctg gtaacgtgga aaactcaaca 5221 gagagaaaag gaagctctct agagagacaa caagcagcca acttagaaga cacaaagagc 5281 tcattggatt gtccagctaa aactgttcta gagtggcagc gtcaaaccca ggactggata 5341 aactctacag aacgccaaga agaaactcgg aaagccccac acaaacaagg ggttggatca 5401 gaggagtccc tggtgcccta cagcaagccc agcttcccgt ctcctggcgg gcacagctca 5461 tcaggaacat cttcctctaa agggtccact ggccctcgga aggctgacgt gctgagaggc 5521 agccaccaga ggaatgccaa tgaccttctc gacattggat atgtgggctc aaatagtcaa 5581 ggacagttta cagagctcga ggagggcaga ggaagtcttc taacatgcgg tgacgtggag 5641 gagaatcccg gccctatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 5701 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 5761 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 5821 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 5881 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 5941 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 6001 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 6061 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 6121 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 6181 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 6241 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 6301 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 6361 gagctgtaca agtagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt 6421 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 6481 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 6541 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 6601 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 6661 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 6721 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 6781 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 6841 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 6901 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 6961 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 7021 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 7081 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa 7141 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 7201 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 7261 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 7321 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 7381 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 7441 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg 7501 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt 7561 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt 7621 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc 7681 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc 7741 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga 7801 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat 7861 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca 7921 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga 7981 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc 8041 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat 8101 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga 8161 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg 8221 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt 8281 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa 8341 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg 8401 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 8461 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc 8521 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 8581 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg 8641 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 8701 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 8761 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 8821 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 8881 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 8941 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 9001 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 9061 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 9121 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 9181 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 9241 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 9301 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 9361 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 9421 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 9481 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 9541 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tttttttgtt 9601 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 9661 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 9721 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 9781 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 9841 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 9901 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 9961 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 10021 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 10081 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 10141 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 10201 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 10261 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 10321 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 10381 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 10441 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 10501 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 10561 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 10621 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 10681 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 10741 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 10801 gacgtc //