LOCUS Exported 7916 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plant expression vector (35S) for direct cloning of artificial microRNAs into Arabidopsis thaliana MIR390a precursor. ACCESSION . VERSION . KEYWORDS pFK210B-AtMIR390a-B/c SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7916) AUTHORS Carbonell A, Takeda A, Fahlgren N, Johnson SC, Cuperus JT, Carrington JC TITLE New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis. JOURNAL Plant Physiol. 2014 Mar 19. PUBMED 24647477 REFERENCE 2 (bases 1 to 7916) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7916 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 1..23 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA (truncated)" terminator 47..299 /label=NOS terminator /note="nopaline synthase terminator and poly(A) signal" CDS complement(319..870) /codon_start=1 /gene="Streptomyces hygroscopicus bar" /product="phosphinothricin acetyltransferase" /label=BlpR /note="confers resistance to bialophos or phosphinothricin" /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF WQLDFSLPVPPRPVLPVTEI" promoter complement(912..1091) /label=NOS promoter /note="nopaline synthase promoter" primer_bind 1328..1350 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 1342..1359 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 1343..1359 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 1369..1388 /label=T7 /note="T7 promoter, forward primer" promoter 1369..1387 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 1412..1428 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 1413..1429 /label=KS primer /note="common sequencing primer, one of multiple similar variants" promoter 1966..2311 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" primer_bind 2254..2274 /label=35S promoter /note="CaMV 35S promoter, forward primer" protein_bind 2336..2360 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter 2654..2684 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 2738..3397 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(2804..2823) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 3739..4044 /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 3923..3942 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(4308..4332) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" terminator 4339..4980 /label=E9 terminator /note="terminator and polyadenylation signal from the pea rbcS-E9 gene" promoter complement(5002..5020) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5041..5057) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5041..5057) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5054..5076) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5065..5081 /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(5089..5119) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5134..5155 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5272..5289) /label=L4440 /note="L4440 vector, forward primer" misc_feature 5394..5418 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" rep_origin complement(5509..6097) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5589..5608) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6268..7056) /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 7347..7782 /label=pSa ori /note="origin of replication from bacterial plasmid pSa" ORIGIN 1 tggcaggata tattgtggtg taacgttatc agcttgcatg ccggtcgatc tagtaacata 61 gatgacaccg cgcgcgataa tttatcctag tttgcgcgct atattttgtt ttctatcgcg 121 tattaaatgt ataattgcgg gactctaatc ataaaaaccc atctcataaa taacgtcatg 181 cattacatgt taattattac atgcttaacg taattcaaca gaaattatat gataatcatc 241 gcaagaccgg caacaggatt caatcttaag aaactttatt gccaaatgtt tgaacgatct 301 gcttgactct aggggtcatc agatttcggt gacgggcagg accggacggg gcggcaccgg 361 caggctgaag tccagctgcc agaaacccac gtcatgccag ttcccgtgct tgaagccggc 421 cgcccgcagc atgccgcggg gggcatatcc gagcgcctcg tgcatgcgca cgctcgggtc 481 gttgggcagc ccgatgacag cgaccacgct cttgaagccc tgtgcctcca gggacttcag 541 caggtgggtg tagagcgtgg agcccagtcc cgtccgctgg tggcgggggg agacgtacac 601 ggtggactcg gccgtccagt cgtaggcgtt gcgtgccttc cagggacccg cgtaggcgat 661 gccggcgacc tcgccgtcca cctcggcgac gagccaggga tagcgctccc gcagacggac 721 gaggtcgtcc gtccactcct gcggttcctg cggctcggta cggaagttga ccgtgcttgt 781 ctcgatgtag tggttgacga tggtgcagac cgccggcatg tccgcctcgg tggcacggcg 841 gatgtcggcc gggcgtcgtt ctgggctcat ggtagatccc ctcgatcgag ttgagagtga 901 atatgagact ctaattggat accgagggga atttatggaa cgtcagtgga gcatttttga 961 caagaaatat ttgctagctg atagtgacct taggcgactt ttgaacgcgc aataatggtt 1021 tctgacgtat gtgcttagct cattaaactc cagaaacccg cggctcagtg gctccttcaa 1081 cgttgcggtt ctgtcagttc caaacgtaaa acggcttgtc ccgcgtcatc ggcgggggtc 1141 ataacgtgac tcccttaatt ctccgctcat gtatcgataa cattaacgtt tacaatttcg 1201 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 1261 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca 1321 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca 1381 ctatagggcg aattgggtac cgggcccccc ctcgaggtcg acggtatcga taagcttgat 1441 atcgaattcc tgcagcccgg gggatccatt cggtccccag attagccttt tcaatttcag 1501 aaagaatgct aacccacaga tggttagaga ggcttacgca gcaggtttca tcaagacgat 1561 ctacccgagc aataatctcc aggaaatcaa ataccttccc aagaaggtta aagatgcagt 1621 caaaagattc aggactaact gcatcaagaa cacagagaaa gatatatttc tcaagatcag 1681 aagtactatt ccagtatgga cgattcaagg cttgcttcac aaaccaaggc aagtaataga 1741 gattggagtc tctaaaaagg tagttcccac tgaatcaaag gccatggagt caaagattca 1801 aatagaggac ctaacagaac tcgccgtaaa gactggcgaa cagttcatac agagtctctt 1861 acgactcaat gacaagaaga aaatcttcgt caacatggtg gagcacgaca cacttgtcta 1921 ctccaaaaat atcaaagata cagtctcaga agaccaaagg gcaattgaga cttttcaaca 1981 aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt 2041 gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc 2101 catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag 2161 catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 2221 ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat 2281 ataaggaagt tcatttcatt tggagagaac acgggggacg agcttctaga ggatcacaag 2341 tttgtacaaa aaagcaggct ccgcggccgc ccccttcacc tatagggggg aaaaaaaggt 2401 agtcatcaga tatatatttt ggtaagaaaa tatagaaatg aataatttca cgtttaacga 2461 agaggagatg acgtgtgttc cttcgaaccc gagttttgtt cgtctataaa tagcaccttc 2521 tcttctcctt cttcctcact tccatctttt tagcttcact atctctctat aatcggtttt 2581 atctttctct aagtcacaac ccaaaaaaac aaagtagaga agaatctgta agagaccatt 2641 aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga ttttgagtta 2701 ggagccgtcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata 2761 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt 2821 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt 2881 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa 2941 tgctcatccg gagttccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt 3001 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga 3061 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg 3121 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa 3181 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc 3241 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc 3301 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt 3361 acaacagtac tgcgatgagt ggcagggcgg ggcgtaaacg cgtggagccg gcttactaaa 3421 agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa tatatactga 3481 tatgtatacc cgaagtatgt caaaaagagg tatgctatga agcagcgtat tacagtgaca 3541 gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc tccggtctgg 3601 taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa 3661 atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct tttgctgacg 3721 agaacagggg ctggtgaaat gcagtttaag gtttacacct ataaaagaga gagccgttat 3781 cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccggccgacg gatggtgatc 3841 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg 3901 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtttcc 3961 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt 4021 aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagcca gtctgcacct 4081 cgacggtctc acattggctc ttcttactac aatgaaaaag gccgaggcaa aacgcctaaa 4141 atcacttgag aatcaattct ttttactgtc catttaagct atcttttata aacgtgtctt 4201 attttctatc tcttttgttt aaactaagaa actatagtat tttgtctaaa acaaaacatg 4261 aaagaacaga ttagatctca tctttagtct caagggtggg cgcgccgacc cagctttctt 4321 gtacaaagtg gtgatcctag ctttcgttcg tatcatcggt ttcgacaacg ttcgtcaagt 4381 tcaatgcatc agtttcattg cgcacacacc agaatcctac tgagtttgag tattatggca 4441 ttgggaaaac tgtttttctt gtaccatttg ttgtgcttgt aatttactgt gttttttatt 4501 cggttttcgc tatcgaactg tgaaatggaa atggatggag aagagttaat gaatgatatg 4561 gtccttttgt tcattctcaa attaatatta tttgtttttt ctcttatttg ttgtgtgttg 4621 aatttgaaat tataagagat atgcaaacat tttgttttga gtaaaaatgt gtcaaatcgt 4681 ggcctctaat gaccgaagtt aatatgagga gtaaaacact tgtagttgta ccattatgct 4741 tattcactag gcaacaaata tattttcaga cctagaaaag ctgcaaatgt tactgaatac 4801 aagtatgtcc tcttgtgttt tagacattta tgaactttcc tttatgtaat tttccagaat 4861 ccttgtcaga ttctaatcat tgctttataa ttatagttat actcatggat ttgtagttga 4921 gtatgaaaat attttttaat gcattttatg acttgccaat tgattgacaa catgcatcaa 4981 tcggagctcc agcttttgtt ccctttagtg agggttaatt ccgagcttgg cgtaatcatg 5041 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 5101 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 5161 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 5221 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 5281 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 5341 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgaagg ccttgacagg 5401 atatattggc gggtaaacta agtcgctgta tgtgtttgtt tgagatctca tgtgagcaaa 5461 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 5521 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 5581 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 5641 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 5701 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 5761 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 5821 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 5881 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 5941 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaagaag 6001 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 6061 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 6121 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 6181 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 6241 tatatatgtg taacattggt ctagtgatta tttgccgact accttggtga tctcgccttt 6301 cacgtagtga acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttgtcc 6361 aagataagcc tgcctagctt caagtatgac gggctgatac tgggccggca ggcgctccat 6421 tgcccagtcg gcagcgacat ccttcggcgc gattttgccg gttactgcgc tgtaccaaat 6481 gcgggacaac gtaagcacta catttcgctc atcgccagcc cagtcgggcg gcgagttcca 6541 tagcgttaag gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg gatcaaagag 6601 ttcctccgcc gctggaccta ccaaggcaac gctatgttct cttgcttttg tcagcaagat 6661 agccagatca atgtcgatcg tggctggctc gaagatacct gcaagaatgt cattgcgctg 6721 ccattctcca aattgcagtt cgcgcttagc tggataacgc cacggaatga tgtcgtcgtg 6781 cacaacaatg gtgacttcta cagcgcggag aatctcgctc tctccagggg aagccgaagt 6841 ttccaaaagg tcgttgatca aagctcgccg cgttgtttca tcaagcctta cggtcaccgt 6901 aaccagcaaa tcaatatcac tgtgtggctt caggccgcca tccactgcgg agccgtacaa 6961 atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg acgccaacta cctctgatag 7021 ttgagtcgat acttcggcga tcaccgcttc cctcataaca ccccttgtat tactgtttat 7081 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca 7141 gagattttga gacacaacgt ggctttgttg aataaatcga acttttgctg agttgaagga 7201 tcagatcacg catcttcccg acaacgcaga ccgttccgtg gcaaagcaaa agttcaaaat 7261 caccaactgg tccacctaca acaaagctct catcaaccgt ggctccctca ctttctggct 7321 ggatgatggg gcgattcagg cgatccccat ccaacagccc gccgtcgagc gggctttttt 7381 atccccggaa gcctgtggat agagggtagt tatccacgtg aaaccgctaa tgccccgcaa 7441 agccttgatt cacggggctt tccggcccgc tccaaaaact atccacgtga aatcgctaat 7501 cagggtacgt gaaatcgcta atcggagtac gtgaaatcgc taataaggtc acgtgaaatc 7561 gctaatcaaa aaggcacgtg agaacgctaa tagccctttc agatcaacag cttgcaaaca 7621 cccctcgctc cggcaagtag ttacagcaag tagtatgttc aattagcttt tcaattatga 7681 atatatatat caattattgg tcgcccttgg cttgtggaca atgcgctacg cgcaccggct 7741 ccgcccgtgg acaaccgcaa gcggttgccc accgtcgagc gccagcgcct ttgcccacaa 7801 cccggcggcc ggccgcaaca gatcgtttta taaatttttt tttttgaaaa agaaaaagcc 7861 cgaaaggcgg caacctctcg ggcttctgga tttccgatcc ccggaattag agatct //