LOCUS Exported 6601 bp ds-DNA circular SYN 26-MAY-2017 DEFINITION Ligation of inverted Fragment 1 into Fragment 2. ACCESSION BI-16_+_RRP6_WT_ VERSION . KEYWORDS pKK-RNAi-nucEGFPmiR-TEV-FLAG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6601) AUTHORS Self JOURNAL Unpublished. REFERENCE 2 (bases 1 to 6601) AUTHORS . TITLE Direct Submission JOURNAL Exported Sunday, May 28, 2017 from SnapGene 3.3.3 http://www.snapgene.com COMMENT SECID/File created by SciEd Central, Scientific & Educational Software SECNOTES|Vector molecule: Fragment 2 Fragment ends: XmaJI and ClaI Fragment size: 7594 Insert molecule: Fragment 1 Fragment ends: XmaJI and ClaI Fragment size: 1494 FEATURES Location/Qualifiers source 1..6601 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 270..543 /note="TK poly(A) signal" misc_feature complement(647..705) /label=Neg miR /note="Neg miR" misc_feature complement(785..843) /label=Neg miR /note="Neg miR" misc_feature complement(923..981) /label=Neg miR /note="Neg miR" misc_feature complement(1052..1137) /note="NLS" CDS complement(1138..1852) /codon_start=1 /note="EGFP" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELY" misc_feature 1861..2388 /note="BI Promoter" protein_bind 1866..1884 /bound_moiety="Tet repressor" /note="tet operator" protein_bind 1887..1905 /bound_moiety="Tet repressor" /note="tet operator" primer_bind 1969..1984 /note="100F" primer_bind 2240..2258 /note="372F" primer_bind 2265..2280 /note="100F" protein_bind 2344..2362 /bound_moiety="Tet repressor" /note="tet operator" protein_bind 2365..2383 /bound_moiety="Tet repressor" /note="tet operator" misc_feature 2413..2433 /label=TEV /note="TEV-L" misc_feature 2474..2494 /label=TEV /note="TEV-R" CDS 2501..2527 /codon_start=1 /note="FLAG" /translation="DYKDDDDK" polyA_signal 2559..2783 /note="BGH poly(A) signal" /note="bovine growth hormone polyadenylation signal" protein_bind 3067..3114 /bound_moiety="FLP recombinase" /note="FRT" CDS 3122..4144 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /note="Hygromycin B" /note="confers resistance to hygromycin" /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE " polyA_signal 4274..4404 /note="SV40 poly(A) signal" /note="SV40 polyadenylation signal" rep_origin complement(4787..5460) /direction=LEFT /note="pUC origin" /note="pUC origin color: #66cc99; direction: LEFT" CDS complement(5605..6465) /codon_start=1 /product="beta-lactamase" /note="Ampicillin" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6466..6564) /gene="bla" /note="Amp promoter" ORIGIN 1 gacggatcgg gagatccgct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc 61 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga 121 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 181 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat 241 tctccgctcc atcgttcaga tccttatcga tgctatggca gggcctgccg ccccgacgtt 301 ggctgcgagc cctgggcctt cacccgaact tggggggtgg ggtggggaaa aggaagaaac 361 gcgggcgtat tggccccaat ggggtcacgg tggggtatcg acagagtgcc agccctggga 421 ccgaaccccg cgtttatgaa caaacgaccc aacacccgtg cgttttattc tgtcttttta 481 ttgccgtcat agcgcgggtt ccttccggta ttgtctcctt ccgtgtttca gttagcctcc 541 cccatcagcg aaccgcgggc cctctagatc actagatatg acgcgtgcgg ccgcagatct 601 gggccatttg ttccatgtga gtgctagtaa caggccttgt gtcctgaaat gtactgcgtg 661 gagacgtcag tcagtggcca aaacgtctcc acgcgcagta catttcagca tacagccttc 721 agcaagcctc caggatctgg gccatttgtt ccatgtgagt gctagtaaca ggccttgtgt 781 cctgaaatgt actgcgtgga gacgtcagtc agtggccaaa acgtctccac gcgcagtaca 841 tttcagcata cagccttcag caagcctcca ggatctgggc catttgttcc atgtgagtgc 901 tagtaacagg ccttgtgtcc tgaaatgtac tgcgtggaga cgtcagtcag tggccaaaac 961 gtctccacgc gcagtacatt tcagcataca gccttcagca agcctccagg atcacttaag 1021 actagcgctc caattgcact acctcctctt actacacctt gcgtttcttc tttgggtcga 1081 cctttctctt ctttttaggg tccaccttcc gctttttctt ggggtctgct gcggccttgt 1141 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt 1201 gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt 1261 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct 1321 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct 1381 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca 1441 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt 1501 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg 1561 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt 1621 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc 1681 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg 1741 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct 1801 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtcctag 1861 gacgatctct atcactgata gggagatctc tatcactgat agggagagct ctgcttatat 1921 agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag ttgttacgac 1981 attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg tcaatggggt 2041 ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg tactgccaaa 2101 accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt 2161 cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa 2221 tagggggcgt acttggcata tgatacactt gatgtactct agaaaaatgt cgtaacaact 2281 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 2341 ctctccctat cagtgataga gatctcccta tcagtgatag agatcgtcaa gcttggtacc 2401 gagctcggat ccgaaaacct gtacttccaa ggaaccggtg gtcacctgta caatcgatcc 2461 tgcaggagct agcgagaatt tgtattttca gggtgatatc gattacaagg atgacgacga 2521 taagtagctc gaggtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc 2581 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 2641 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 2701 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 2761 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 2821 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 2881 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 2941 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 3001 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 3061 tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc 3121 caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 3181 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 3241 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 3301 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 3361 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 3421 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc 3481 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 3541 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 3601 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 3661 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 3721 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 3781 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 3841 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg 3901 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 3961 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 4021 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 4081 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga 4141 atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 4201 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 4261 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 4321 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 4381 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 4441 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 4501 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 4561 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 4621 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 4681 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 4741 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 4801 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 4861 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 4921 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 4981 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 5041 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 5101 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 5161 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 5221 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 5281 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 5341 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 5401 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 5461 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 5521 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 5581 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 5641 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 5701 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 5761 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 5821 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 5881 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 5941 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 6001 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 6061 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 6121 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 6181 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 6241 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 6301 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 6361 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 6421 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 6481 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 6541 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 6601 c //