LOCUS Exported 8328 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS MIGS4.2 (FF575) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8328) AUTHORS de Felippes FF, Wang JW, Weigel D TITLE MIGS: MiRNA Induced Gene Silencing. JOURNAL Plant J. 2011 Dec 28. doi: 10.1111/j.1365-313X.2011.04896.x. PUBMED 22211571 REFERENCE 2 (bases 1 to 8328) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8328 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 542..564 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA (truncated)" terminator 588..840 /label=NOS terminator /note="nopaline synthase terminator and poly(A) signal" CDS complement(860..1654) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERGRTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTQGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(971..990) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 1581..1600 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter 1840..1858 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 1840..1857 /label=SP6 /note="SP6 promoter, forward primer" terminator 1891..2598 /label=OCS terminator /note="octopine synthase terminator" primer_bind 3507..3523 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 3508..3524 /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3558..3574) /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3558..3574) /label=pBluescriptSK /note="For pBluescript vector" protein_bind 3580..3704 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 3729..3759 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 3813..4472 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(3879..3898) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 4814..5119 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 4998..5017 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(5160..5284) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" terminator 5291..5932 /label=E9 terminator /note="terminator and polyadenylation signal from the pea rbcS-E9 gene" promoter complement(5954..5972) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5993..6009) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5993..6009) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6006..6028) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6017..6033 /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(6041..6072) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6087..6108 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6225..6242) /label=L4440 /note="L4440 vector, forward primer" misc_feature 6347..6371 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" rep_origin complement(6462..7050) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6542..6561) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7221..8009) /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK" rep_origin 8300..407 /label=pSa ori /note="origin of replication from bacterial plasmid pSa" ORIGIN 1 tttttatccc cggaagcctg tggatagagg gtagttatcc acgtgaaacc gctaatgccc 61 cgcaaagcct tgattcacgg ggctttccgg cccgctccaa aaactatcca cgtgaaatcg 121 ctaatcaggg tacgtgaaat cgctaatcgg agtacgtgaa atcgctaata aggtcacgtg 181 aaatcgctaa tcaaaaaggc acgtgagaac gctaatagcc ctttcagatc aacagcttgc 241 aaacacccct cgctccggca agtagttaca gcaagtagta tgttcaatta gcttttcaat 301 tatgaatata tatatcaatt attggtcgcc cttggcttgt ggacaatgcg ctacgcgcac 361 cggctccgcc cgtggacaac cgcaagcggt tgcccaccgt cgagcgccag cgcctttgcc 421 cacaacccgg cggccggccg caacagatcg ttttataaat tttttttttt gaaaaagaaa 481 aagcccgaaa ggcggcaacc tctcgggctt ctggatttcc gatccccgga attagagatc 541 ttggcaggat atattgtggt gtaacgttat cagcttgcat gccggtcgat ctagtaacat 601 agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc 661 gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat 721 gcattacatg ttaattatta catgcttaac gtaattcaac agaaattata tgataatcat 781 cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc 841 tgcttgactc taggggtcat cagaagaact cgtcaagaag gcgatagaag gcgatgcgct 901 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa 961 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca 1021 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc 1081 aggcatcgcc ctgggtcacg acgagatcct cgccgtcggg catccgcgcc ttgagcctgg 1141 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa 1201 gaccggcttc catccgagta cgtcctcgct cgatgcgatg tttcgcttgg tggtcgaatg 1261 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt 1321 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca 1381 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg 1441 tggccagcca cgatagccgc gctgcctcgt cttggagttc attcagggca ccggacaggt 1501 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag 1561 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg 1621 gagaacctgc gtgcaatcca tcttgttcaa tcatgcctcg atcgagttga gagtgaatat 1681 gagactctaa ttggataccg aggggaattt atggaacgtc agtggagcat ttttgacaag 1741 aaatatttgc tagctgatag tgaccttagg cgacttttga acgcgcaata atggtttctg 1801 acgtatgtgc ttagctcatt aaactccaga aacccgcgga tttaggtgac actatagaat 1861 atcactagta agctagcttg catgccggtc ctgctgagcc tcgacatgtt gtcgcaaaat 1921 tcgccctgga cccgcccaac gatttgtcgt cactgtcaag gtttgacctg cacttcattt 1981 ggggcccaca tacaccaaaa aaatgctgca taattctcgg ggcagcaagt cggttacccg 2041 gccgccgtgc tggaccgggt tgaatggtgc ccgtaacttt cggtagagcg gacggccaat 2101 actcaacttc aaggaatctc acccatgcgc gccggcgggg aaccggagtt cccttcagtg 2161 agcgttatta gttcgccgct cggtgtgtcg tagatactag cccctggggc acttttgaaa 2221 tttgaataag atttatgtaa tcagtctttt aggtttgacc ggttctgccg ctttttttaa 2281 aattggattt gtaataataa aacgcaattg tttgttattg tggcgctcta tcatagatgt 2341 cgctataaac ctattcagca caatatattg ttttcatttt aatattgtac atataagtag 2401 tagggtacaa tcagtaaatt gaacggagaa tattattcat aaaaatacga tagtaacggg 2461 tgatatattc attagaatga accgaaaccg gcggtaagga tctgagctac acatgctcag 2521 gttttttaca acgtgcacaa cagaattgaa agcaaatatc atgcgatcat aggcgtctcg 2581 catatctcat taaagcaggg atcccgcatc agatctatca aacacataga tttacgaaga 2641 tctctaacat taaatcatca gagagataag tttagggagc aagctctttc gcttacacag 2701 agaatcacag aggaaagaga ctttaagaaa actacaactt ttttgaccac tgtgatttct 2761 ctctgcaagc gaaagtactt aatcaccaac acagtagatt gatatatttg ggtttatttg 2821 tgagaagaag aattcactag tggatcccgc actcgagtgt taatcagaaa aactcagatt 2881 aatctacaaa ttcgatcgca caaactagaa actaacacca gatctagata gaaatcacaa 2941 atcgaagagt aattattcga caaaactcaa attatttgaa caaatcggat gatatctatg 3001 aaaccctaat cgagaattaa gatgatatct aacgatcaaa cccagaaaat cgtcttcgat 3061 ctaagattaa cagaatctaa accaaagaac atatacgaaa ttgggatcga acgaaaacaa 3121 aatcgaagat tttgagagaa taaggaacac agaaatttac cttgatcacg gtagagagaa 3181 ttgagagaaa gtttttaaga ttttgagaaa ttgaaatctg aattgtgaag aagaagagct 3241 ctttgggtat tgttttatag aagaagaaga agaaaagacg aggacgacta ggtcacgaga 3301 aagctaaggc ggtgaagcaa tagctaataa taaaatgaca cgtgtattga gcgttgttta 3361 cacgcaaagt tgtttttggc taattgcctt atttttaggt tgaggaaaag tatttgtgct 3421 ttgagttgat aaacacgact cgtgtgtgcc ggctgcaacc actttgacgc cgtttattac 3481 tgactcgtcg ggtaccgggc cccccctcga ggtcgacggt atcgataagc ttgatatcga 3541 attcctgcag cccgggggat ccactagttc tagaggatca caagtttgta caaaaaagct 3601 gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa 3661 cagactacat aatactgtaa aacacaacat atccagtcat attggcggcc ccattaggca 3721 ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg agttaggagc 3781 cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca 3841 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc 3901 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga 3961 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc 4021 atccggagtt ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc 4081 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc 4141 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa 4201 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct 4261 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg 4321 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc 4381 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac 4441 agtactgcga tgagtggcag ggcggggcgt aaacgcgtgg agccggctta ctaaaagcca 4501 gataacagta tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt 4561 atacccgaag tatgtcaaaa agaggtatgc tatgaagcag cgtattacag tgacagttga 4621 cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc 4681 acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag 4741 gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac 4801 aggggctggt gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct 4861 gtttgtggat gtacagagtg atattattga cacgcccggc cgacggatgg tgatccccct 4921 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat 4981 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat 5041 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct 5101 gatgttctgg ggaatataaa tgtcaggctc ccttatacac agccagtctg cacctcgacc 5161 atagtgactg gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct 5221 aatttaatat attgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag 5281 tggtgatcct agctttcgtt cgtatcatcg gtttcgacaa cgttcgtcaa gttcaatgca 5341 tcagtttcat tgcgcacaca ccagaatcct actgagtttg agtattatgg cattgggaaa 5401 actgtttttc ttgtaccatt tgttgtgctt gtaatttact gtgtttttta ttcggttttc 5461 gctatcgaac tgtgaaatgg aaatggatgg agaagagtta atgaatgata tggtcctttt 5521 gttcattctc aaattaatat tatttgtttt ttctcttatt tgttgtgtgt tgaatttgaa 5581 attataagag atatgcaaac attttgtttt gagtaaaaat gtgtcaaatc gtggcctcta 5641 atgaccgaag ttaatatgag gagtaaaaca cttgtagttg taccattatg cttattcact 5701 aggcaacaaa tatattttca gacctagaaa agctgcaaat gttactgaat acaagtatgt 5761 cctcttgtgt tttagacatt tatgaacttt cctttatgta attttccaga atccttgtca 5821 gattctaatc attgctttat aattatagtt atactcatgg atttgtagtt gagtatgaaa 5881 atatttttta atgcatttta tgacttgcca attgattgac aacatgcatc aatcggagct 5941 ccagcttttg ttccctttag tgagggttaa ttccgagctt ggcgtaatca tggtcatagc 6001 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaaghc 6061 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 6121 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 6181 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 6241 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 6301 ttatccacag aatcagggga taacgcagga aagaacatga aggccttgac aggatatatt 6361 ggcgggtaaa ctaagtcgct gtatgtgttt gtttgagatc tcatgtgagc aaaaggccag 6421 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 6481 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 6541 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 6601 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 6661 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 6721 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 6781 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 6841 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 6901 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaag aagagttggt 6961 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 7021 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 7081 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 7141 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 7201 gtgtaacatt ggtctagtga ttatttgccg actaccttgg tgatctcgcc tttcacgtag 7261 tgaacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttg tccaagataa 7321 gcctgcctag cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag 7381 tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac 7441 aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt 7501 aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc 7561 gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga 7621 tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct 7681 ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca 7741 atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa 7801 aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc 7861 aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg 7921 gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc 7981 gatacttcgg cgatcaccgc ttccctcata acaccccttg tattactgtt tatgtaagca 8041 gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt 8101 tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa ggatcagatc 8161 acgcatcttc ccgacaacgc agaccgttcc gtggcaaagc aaaagttcaa aatcaccaac 8221 tggtccacct acaacaaagc tctcatcaac cgtggctccc tcactttctg gctggatgat 8281 ggggcgattc aggcgatccc catccaacag cccgccgtcg agcgggct //